Engineered phloem mobility elements

Abstract

The present disclosure provides methods and compositions for transporting a cargo RNA to the meristem of a plant. More specifically, the present disclosure provides engineered phloem mobility elements that are linked to an RNA cargo and increase transport of the cargo within a plant when provided to the plant, for example, via an Agrobacterium hairy root system. Also provided are methods of use, vectors, nucleic acids, kits, and resulting compositions.

Description

Attorney Docket No.: 16536-20022.40ENGINEERED PHLOEM MOBILITY ELEMENTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 749,330, filed January 24, 2025, entitled “ENGINEERED PHLOEM MOBILITY ELEMENTS,” which is herein incorporated by reference in its entirety for all purposes.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (165362002240SEQLIST.xml; Size: 719,813 bytes; and Date of Creation: January 22, 2026) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] In some aspects, the present invention relates to compositions and methods for transporting an RNA cargo to the meristem of a plant via engineered phloem mobility elements. In some aspects, the engineered phloem mobility elements comprise RNAs with secondary structures that are maintained when the engineered phloem mobility elements are linked to the RNA cargo. In some aspects, engineered phloem mobility elements of the invention relates to methods for gene editing in plants, such as methods that use virus-mediated delivery of guide RNAs for Cas enzymes, wherein the guide RNAs and / or the nucleic acid encoding the Cas enzyme are linked to an engineered phloem mobility element and transported from the root of the plant to the meristem of the plant.BACKGROUND

[0004] There is a need for methods and compositions for increasing or facilitating the transport of RNA cargo within a plant. Transport of biomolecules and other compounds within a plant occurs in the xylem and / or the phloem of the plant, and is tightly regulated by the plant. In some cases, it may be desirable to provide an RNA cargo to the meristem of a plant without contacting the meristem of the plant directly. This disclosure answers this need by providing a system based on engineered tRNA-like mobility elements, which may be linked to an RNA cargo and provided to the plant via a hairy root system.1MF-365821254Attorney Docket No.: 16536-20022.40SUMMARY

[0005] In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:[Structure I] when linked to an RNA cargo, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:(((((((((((((•((((((•( )•)))))) )))))•((( )))•))))))))•• [Structure II] when linked to an RNA cargo, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:[Structure III] when linked to an RNA cargo, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:[Structure IV] when linked to an RNA cargo, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant.

[0006] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising: a) a first stem of 9 nucleotides, b) a first stem loop structure comprising i) a second stem of five nucleotides comprising a bulge comprising one unpaired nucleotide and ii) a first loop comprising four nucleotides, c) a second stem loop structure comprising i) a third stem of five nucleotides and ii) a second loop comprising seven nucleotides, and d) a third stem loop structure comprising i) a third stem of four nucleotides and ii) a third loop comprising four nucleotides, further comprising: e) at least five unpaired nucleotides 5’ of the first stem, f) two unpaired nucleotides between the first stem and the first stem loop structure, g) one unpaired nucleotide between the second stem loop structure and the third stem loop structure, h) eight unpaired nucleotides between the third stem loop structure and the stem, and i) at least four unpaired nucleotides 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant.

[0007] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure2MF-365821254Attorney Docket No.: 16536-20022.40comprising: a) a first stem of eight nucleotides, b) a first stem loop structure comprising i) a second stem of twelve nucleotides, comprising a first bulge comprising one unpaired nucleotide, a second bulge comprising one unpaired nucleotide, a third bulge comprising one unpaired nucleotide, and a first loop comprising six unpaired nucleotides, and ii) a second loop comprising seven nucleotides; and c) a second stem loop structure comprising i) a third stem of three nucleotides and ii) a third loop comprising seven nucleotides, further comprising: d) at least six unpaired nucleotides 5’ of the first stem, e) one unpaired nucleotide between the first stem loop structure and the second stem loop structure, f) one unpaired nucleotide between the second stem loop structure and the first stem, and g) at least two unpaired nucleotides 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

[0008] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising: a) a first stem of six nucleotides, b) a first stem loop structure comprising i) a second stem of four nucleotides, and ii) a first loop comprising nine nucleotides; c) a second stem loop structure comprising i) a third stem of five nucleotides, and ii) a second loop comprising seven nucleotides; and d) a third stem loop structure comprising i) a fourth stem of six nucleotides, and ii) a third loop comprising five nucleotides, further comprising: e) three unpaired nucleotides between the first stem and the first stem loop structure, f) one unpaired nucleotide between the first stem loop structure and the second stem loop structure, g) five unpaired nucleotides between the second stem loop structure and the third stem loop structure, h) one unpaired nucleotides between the third stem loop structure and the first stem, and i) at least one unpaired nucleotide 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

[0009] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising: a) a first stem of five nucleotides, comprising a first bulge comprising one unpaired nucleotide and a second bulge comprising one unpaired nucleotide; b) a first stem loop structure comprising i) a second stem of nine nucleotides, comprising a first bulge comprising three unpaired nucleotides, a second bulge comprising two unpaired nucleotides, a third bulge comprising two unpaired nucleotides, and a fourth bulge comprising three unpaired nucleotides, and ii) a first loop comprising four nucleotides; and c) a second stem loop structure comprising i) a third stem of eight nucleotides, comprising a first bulge comprising one unpaired nucleotide and a second bulge comprising one unpaired nucleotide, and ii) a second 3MF-365821254Attorney Docket No.: 16536-20022.40loop comprising four nucleotides, further comprising: d) three unpaired nucleotides between the first stem and the first stem loop structure, e) four unpaired nucleotides between the first stem loop structure and the second stem loop structure, f) one unpaired nucleotide between the second stem loop structure and the stem, and g) at least one unpaired nucleotide 3’ of the stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

[0010] In some embodiments, the engineered phloem mobility element comprises between 70-85 nucleotides. In some embodiments, the engineered phloem mobility element comprises between 74-82 nucleotides. In some embodiments, the engineered phloem mobility element folds into the secondary structure with an energy partition function of less than -25 EpF. In some embodiments, the engineered phloem mobility element folds into the secondary structure with an energy partition function of less than -30 EpF. In some embodiments, the engineered phloem mobility element folds into the secondary structure with an energy partition function of less than -35 EpF.

[0011] In some embodiments, the engineered phloem mobility element: a) comprises a secondary structure of Structure I, and comprises a sequence selected from the group consisting of SEQ ID NOs: 1-595; b) comprises a secondary structure of Structure II, and comprises a sequence selected from the group consisting of SEQ ID NOs: 596-655; c) comprises a secondary structure of Structure III, and comprises a sequence selected from the group consisting of SEQ ID NOs: 656-690; or d) comprises a secondary structure of Structure IV, and comprises a sequence selected from the group consisting of SEQ ID NOs: 691-752. In some embodiments, the engineered phloem mobility element comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to any one of SEQ ID NO: 1-752, wherein the sequence is not a naturally occurring sequence within the plant. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 370 and 593. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 648 and 653. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 688 and 689. In some embodiments, transport of the linked RNA cargo within the plant is increased compared to transport of the RNA cargo without the engineered phloem mobility element.

[0012] In some embodiments, provided herein is an RNA molecule comprising the engineered phloem mobility element of any one of the preceding embodiments linked to the RNA cargo. In some embodiments, the engineered phloem mobility element is located 3’ to the RNA. In 4MF-365821254Attorney Docket No.: 16536-20022.40some embodiments, the engineered phloem mobility element is located 5’ to the RNA cargo. In some embodiments, the RNA cargo is an mRNA molecule. In some embodiments, the RNA cargo encodes a Cas and / or comprises a guide RNA (gRNA). In some embodiments, the RNA cargo encodes a reporter, optionally a fluorescent reporter. In some embodiments, the reporter is a GFP or an eGFP variant. In some embodiments, provided herein is a nucleic acid encoding the RNA molecule of any one of the preceding embodiments. In some embodiments, provided herein is a vector comprising the nucleic acid of the preceding embodiment. In some embodiments, the RNA cargo is mRNA.

[0013] In some embodiments, provided herein is a plant comprising the RNA molecule of the preceding embodiment. In some embodiments, provided herein is a plant comprising the nucleic acid of the preceding embodiments. In some embodiments, the plant is selected from soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana, cotton, or wheat.

[0014] In some aspects, provided herein is a method of transporting an RNA cargo to a meristem cell of a plant comprising providing to the plant the RNA cargo linked to the engineered phloem mobility element of any of the preceding embodiments. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the plant via a T-DNA vector. In some embodiments, the T-DNA vector is delivered to a root of the plant. In some embodiments, the T-DNA vector comprises a Ri plasmid from Agrobacterium rhizogenes. In some embodiments, the RNA cargo linked to the phloem mobility element is delivered to the root of the plant by an Agrobacterium rhizogenes transformation. In some embodiments, the Agrobacterium rhizogenes transformation produces transgenic hairy roots.

[0015] In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to a root of the plant by injecting a composition comprising the RNA cargo linked to the engineered phloem mobility element into the root. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the plant root by incubating the root with a composition comprising the RNA cargo linked to the engineered phloem mobility element. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the plant root by an Agrobacterium rhizogenes transformation. In some embodiments, the RNA cargo is transported by the plant vascular system when operably linked to the engineered phloem mobility element. In some embodiments, the RNA cargo is transported through the xylem or the phloem when linked to the engineered phloem mobility element.5MF-365821254Attorney Docket No.: 16536-20022.40

[0016] In some embodiments, the RNA cargo comprises nucleic acid encoding a Cas nuclease. In some embodiments, a nucleic acid encoding a gRNA for the Cas nuclease is provided separately to the plant. In some embodiments, the nucleic acid encoding the gRNA for the Cas nuclease is linked to a second phloem mobility element. In some embodiments, the second phloem mobility element is an engineered phloem mobility element of any of the preceding embodiments. In some embodiments, the second phloem mobility element comprises or is derived from: i. a Flower Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or ii. an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop. In some embodiments, the RNA cargo comprises nucleic acid encoding a gRNA for a Cas nuclease. In some embodiments, the Cas nuclease is constitutively expressed in the plant or in the roots of the plant.

[0017] In some embodiments, the plant comprises a rootstock and a scion grafted onto the rootstock. In some embodiments, nucleic acid encoding the Cas nuclease is expressed in the rootstock, wherein the nucleic acid encoding the Cas nuclease is linked to a second phloem mobility element. In some embodiments, the second phloem mobility element is an engineered phloem mobility element of any one of the preceding embodiments. In some embodiments, the second phloem mobility element comprises or is derived from: i. a Flower Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or ii. an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.

[0018] In some embodiments, the RNA cargo comprises i) nucleic acid encoding a Cas nuclease and ii) a gRNA for the Cas nuclease. In some embodiments, the nucleic acid encoding the Cas nuclease is transported to the meristem, wherein the Cas nuclease is translated in the meristem. In some embodiments, a genomic target within a cell in the meristem is edited. In some embodiments, the plant comprises a rootstock and a scion grafted onto the rootstock. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and / or rootstock is a dicot. In some embodiments, the plant is a dicot. In some embodiments, the scion and / or rootstock is a monocot. In some embodiments, the plant is a monocot. In some embodiments, the rootstock and / or scion, or plant is soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana, or wheat. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in dicots, optionally wherein the nucleic acid encoding the Cas nuclease is codon-optimized for expression in soybean. In some 6MF-365821254Attorney Docket No.: 16536-20022.40embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in monocots. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in corn, soy, or wheat. In some embodiments, the method further comprises retrieving a progeny of the plant, wherein the progeny has an altered genome.

[0019] In some embodiments, the guide RNA further comprises: (a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide RNA; or (b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide RNA; or (c) both (a) and (b); wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar. In some embodiments, each of the one or more modified nucleotides is independently selected from the group consisting of 2’-O-methyl nucleotide, a 2’-0-methyl-3’phosphorothioate nucleotide, a 2’-O-methyl-3’phosphonoacetate nucleotide, and a 2’-0-methyl-3’-phosphonothioacetate nucleotide. In some embodiments, the one or more modified nucleotides comprises a modified intemucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group. In some embodiments, the gRNA comprises a sequence that is heterologous to the plant.

[0020] In some embodiments, the method further comprises delivering a donor template DNA to the plant. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the plant at the genomic target. In some embodiments, the meristem cell is in a shoot apical meristem or an axillary meristem. In some embodiments, the editing of the genomic target results in the increased expression of a gene of interest in the plant, wherein the genomic target inhibits the gene of interest when expressed in a control plant. In some embodiments, the genomic target is involved in viral defense, Non-Homologous End Joining (NHEJ), Mismatch Repair (MMR), or condensing chromatin. In some embodiments, the method further comprises screening the plant for successful editing of the genomic target, said screening comprising: a) visually assessing the plant for at least one desired phenotype; and / or b) sequencing nucleic acid of cells produced by the meristem cell.

[0021] In some embodiments, provided herein is a plant produced by the method of the preceding embodiment, wherein the produced plant comprises the edited genomic target. In some embodiments, provided herein is a seed produced by the method of the preceding embodiment, wherein the produced seed comprises the edited genomic target. In some embodiments, the meristem cell comprises the edited genomic target. In some embodiments,7MF-365821254Attorney Docket No.: 16536-20022.40provided herein is a kit comprising the viral vector system of the preceding embodiment and an instruction manual for using the kit.

[0022] In some aspects, provided herein is a method for assessing the ability of a candidate engineered phloem mobility element to promote mobility of an RNA cargo within a plant, comprising: a) delivering the candidate engineered phloem mobility element linked to the RNA cargo to the roots of the plant via an Agrobacterium rhizogenes vector, wherein the engineered phloem mobility element comprises a secondary structure when linked to the RNA cargo, and b) measuring the amount of the RNA cargo in the meristem of the plant. In some embodiments, the RNA cargo comprises mRNA. In some embodiments, the RNA cargo comprises a reporter system.

[0023] In some aspects, provided herein is a rootstock comprising a nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element comprising a secondary structure of:[Structure I], wherein the engineered phloem mobility element is not a naturally occurring RNA in the plant. In some aspects, provided herein is a rootstock comprising a nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element comprising a secondary structure of: > (((((((((((((•((((((•( )•)))))) )))))•((( )))•))))))))•• [Structure II], wherein the engineered phloem mobility element is not a naturally occurring RNA in the plant. In some aspects, provided herein is a rootstock comprising a nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element comprising a secondary structure of:[Structure III], wherein the engineered phloem mobility element is not a naturally occurring RNA in the plant. In some aspects, provided herein is a rootstock comprising a nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element comprising a secondary structure of:[Structure IV], wherein the engineered phloem mobility element is not a naturally occurring RNA in the plant.8MF-365821254Attorney Docket No.: 16536-20022.40BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG.1 shows a protocol for assessing candidate engineered phloem mobility elements for transporting RNA cargo in planta.

[0025] FIGs.2A-2D show dot-bracket notation of the secondary structures of the engineered phloem mobility elements comprising the structures of Structure I (RMS1, FIG. 2A), Structure II (RMS2, FIG. 2B), Structure III (RMS3, FIG. 2C), and Structure IV (RMS4, FIG. 2D).

[0026] FIGs. 3A-3D shows representations of the secondary structures of examples of engineered phloem mobility elements. FIG. 3A shows an example secondary structure of Structure I (SEQ ID NO: 1). FIG. 3B shows an example secondary structure of Structure II (SEQ ID NO: 596). FIG. 3C shows an example secondary structure of Structure III (SEQ ID NO: 656). FIG.3D shows an example secondary structure of Structure IV (SEQ ID NO: 691).

[0027] FIG. 4 shows the number of Cas molecules detected in the shoots of plants wherein the Cas enzyme is the cargo RNA linked to the engineered phloem mobility element and delivered to the root of the plant. All constructs tested showed increase mobility of Cas into the shoots of the plant compared to the negative control.

[0028] FIGs.5A-5C show the percent editing in shoots for each guide RNA construct tested.FIG. 5A shows the percent of edited shoots generated with guide RNA CR980 (SEQ ID NO: 653). FIG. 5B shows the percent of edited shoots generated with guide RNA CR1808 (SEQ ID NO: 654). FIG. 5C shows the percent of edited shoots generated with guide RNA CR423 (SEQ ID NO: 655).DETAILED DESCRIPTION

[0029] All references cited herein are hereby incorporated by reference in their entirety.I. Definitions

[0030] The use of the terms “a” and “an” and “the” and “at least one” and similar language in the context of describing embodiments of the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context.9MF-365821254Attorney Docket No.: 16536-20022.40The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0031] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism.

[0032] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0033] As used herein, the phrase “codon optimization” refers to the process of modifying a nucleic acid sequence for use in a desired host kingdom, phylum, class, order, family, genus, or species, by replacing at least one codon of the nucleic acid with codons that are more frequently used in the genes of the desired host kingdom, phylum, class, order, family, genus, or species, without alteration of the amino acid sequence encoded by the nucleic acid.

[0034] As used herein, the term “complementary” refers to sequences with at least sufficient complementarity to permit enough base-paring for two nucleic acids to hybridize (for example, for a tether to hybridize with or bind to a gRNA or donor DNA), which in some examples may be under typical physiological conditions for the cell. In some examples, the oligonucleotide or polynucleotide is at least 80% complementary to the target, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target. Given the complementary pairings of nucleotide bases, the nucleotide sequences of the present disclosure should be understood to include their complementary sequences.

[0035] As used herein, the term “complex” refers to two or more associated components, such as two or more associated nucleic acids and / or proteins. A complex may include two or more covalently linked nucleic acids and / or proteins, two or more non-covalently linked nucleic acids and / or proteins, or a combination thereof.

[0036] As used herein, the terms “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” can be interchanged and are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0037] As used herein, the term “CRISPR-Cas nuclease,” “CRISPR Cas nuclease,” and “Cas nuclease” are used interchangeably herein to refer to RNA-directed nucleases and RNA-guided nucleases.10MF-365821254Attorney Docket No.: 16536-20022.40

[0038] As used herein, the term “endogenous” refers to something that can be found in an organism prior to human intervention. An “endogenous sequence” refers to a DNA sequence located in the genome of the organism prior to editing.

[0039] As used herein, the term “engineered” means artificial, synthetic, or not occurring in nature. For example, a polynucleotide that includes two DNA sequences that are heterologous to each other can be engineered or synthesized by recombinant nucleic acid techniques.

[0040] As used herein, the term “exogenous” refers to something that cannot be found in an organism prior to human intervention. An “exogenous sequence” refers to a DNA sequence that is not located in the genome of an organism prior to editing. An exogenous sequence can be an edited sequence, a synthetic sequence, or a sequence from a different organism.

[0041] As used herein, the terms “heritable genetic modification”, “heritable edit”, and “heritable modification” refer to any insertion, substitution, or deletion in the genomic sequence of a plant that is at least present in a meristem cell of the plant, such that at least one progeny of the plant possesses the same altered genomic sequence.

[0042] As used herein, the terms “a graft,” “to graft,” and “grafting” refer to the technique wherein two plants are joined by their vasculature such that they fuse to form a single grafted plant. The plant that maintains or will maintain the root system after grafting is referred to herein as the “rootstock”. The plant grafted onto the rootstock is referred to herein as the “shoot”, “plant scion” or “scion”. Grafting includes “micrografting” (Pena et al. Plant Cell Rep 1995, 14: 616-619; CN105519434A; CN110178564A), “minigrafting” (Marques et al. Sci Hortic 2011, 129: 176-182), and other forms of grafting known to those in the art.

[0043] As used herein, the term “heterograft” refers to a graft between a rootstock and a scion of different species.

[0044] As used herein, the term “homograft” refers to a graft between a rootstock and a scion of the same species.

[0045] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0046] As used herein, the terms “modification,” “edit”, and “modify” are used interchangeably herein to refer to any insertion, substitution, or deletion of any number of nucleotides in a genomic sequence.

[0047] As used herein, the term “mobile” refers to the ability of a molecule or a collection of molecules to move within the plant. A nucleic acid encoding a Cas nuclease linked to an engineered phloem mobility element results in a mobile Cas, which is capable of being 11MF-365821254Attorney Docket No.: 16536-20022.40transported through the plant vascular system to the meristem of the plant, including through a graft junction and then being translated in the meristem. Similarly, a linkage of an RNA molecule and an engineered phloem mobility element results in a “mobile RNA”, which is capable of being transported through the plant vascular system to the meristem of the plant, including through a graft junction.

[0048] As used herein, the term “linked” means connected, for example via a covalent bond. For example, an engineered phloem mobility element and a cargo are “linked” when they are elements of a single RNA that can be transported from one tissue to the meristem.

[0049] As used herein, the terms “orthologous,” “ortholog,” or “orthologue” are used to describe genes or the RNAs or proteins encoded by those genes that are from different species but which have the same function (e.g., encode RNAs which exhibit the same meristem transport function). Orthologous genes will typically encode RNAs or proteins with some degree of sequence identity and can also exhibit conservation of sequence motifs, and / or conservation of structural features including RNA stem loop structures.

[0050] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.

[0051] As used herein, the phrase “substantially purified” defines an isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition. The phrase “substantially purified RNA molecule” is used herein to describe an RNA molecule 12MF-365821254Attorney Docket No.: 16536-20022.40which has been separated from other contaminant compounds including, but not limited to polypeptides, lipids, and carbohydrates. In certain embodiments, a substantially purified RNA is at least 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% free of contaminating compounds by weight. A substantially purified RNA molecule can be combined with other compounds including buffers, RNase inhibitors, surfactants, and the like in a composition.

[0052] As used herein, the term “polynucleotide” refers to a nucleic acid molecule containing multiple nucleotides and encompasses both “oligonucleotides” (defined here as a polynucleotide molecule of between 2-25 nucleotides in length) and polynucleotides of 26 or more nucleotides. Polynucleotides are generally described as single- or double-stranded. Where a polynucleotide contains double- stranded regions formed by intra- or intermolecular hybridization, the length of each double- stranded region is conveniently described in terms of the number of base pairs. Aspects of this invention include the use of polynucleotides or compositions containing polynucleotides; embodiments include one or more oligonucleotides or polynucleotides or a mixture of both, including single- or double-stranded RNA or single-or double- stranded DNA or double- stranded DNA / RNA hybrids or chemically modified analogues or a mixture thereof. In various embodiments, a polynucleotide includes a combination of ribonucleotides and deoxyribonucleotides (e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides), or includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In embodiments, the polynucleotide includes chemically modified nucleotides (see, e.g., Verma and Eckstein Annu. Rev. Biochem. 1998, 67: 99-134); for example, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications; modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis; and oligonucleotides or polynucleotides can be labelled with a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels) or other label (e.g., biotin or an isotope). Modified nucleic acids, particularly modified RNAs, are disclosed in U.S. Pat. No. 9,464,124, incorporated by reference in its entirety herein.

[0053] As used herein, the terms “progeny” or “plant progeny” refer to any zygote, embryo, endosperm, callus, seed, seedling, or second generation of a plant that is produced after a parent plant cell undergoes meiosis and, in some cases, syngamy.13MF-365821254Attorney Docket No.: 16536-20022.40

[0054] As used herein, the phrase “sequence identity” refers to the percent similarity of two polynucleotides or polypeptides. A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available at ncbi[dot]nlm[dot]nih[dot]gov / BLAST. See, e.g., Altschul et al. Mol. Biol. 1990, 215:403-410. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol., 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See Mol. Biol., 48: 443-453 (1970).

[0055] As used herein, the phrase “T-DNA” or “transfer DNA” refer to the DNA transferred from the tumor-inducing plasmid of species of bacteria such as but not limited to Agrobacterium tumefaciens and Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), to the nuclear genome of a host plant.

[0056] As used herein, the phrase “T-DNA vector” refers to a transfer DNA vector system comprising as least a disarmed tumor inducing (Ti) plasmid of species of bacteria such as, but not limited to, Agrobacterium tumefaciens and Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), containing a T-DNA and a vector backbone, and a helper plasmid containing vir virulence genes. A T-DNA vector system may be a binary vector system; a superbinary vector system wherein the Ti plasmid also comprises virulence genes (Komari et al. Plant Physiol 2007, 145(4): 1155-1160); or a ternary vector system wherein the system further comprises an accessory plasmid or virulence helper plasmid comprising an additional virulence gene cluster (Anand et al. Plant Mol Biol 2018, 97(1-2): 187-200).

[0057] As used herein, the terms “template,” “template sequence,” “donor template,” and “donor template sequence,” can all be used to refer to a DNA polynucleotide provided to a nucleus, cell, or plant in combination with other genome editing reagents in order to integrate a DNA sequence from the DNA polynucleotide into the genome of the nucleus, cell, or plant.14MF-365821254Attorney Docket No.: 16536-20022.40

[0058] As used herein, the terms “vascular system” or “vasculature” refer to the transport systems within the plant. This includes xylem, phloem, and cambium.

[0059] As used herein, the term “engineered” means modified in some way by human intervention.

[0060] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art.

[0061] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0062] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.II. Engineered Phloem Mobility Elements

[0063] In some aspects, the present application provides engineered phloem mobility elements. In some embodiments, the engineered phloem mobility elements provided herein are linked to an RNA cargo and delivered to a plant. In some embodiments, the engineered phloem mobility elements comprise RNA with a secondary structure that is maintained when linked to a larger RNA molecule, for example, an RNA cargo. In some embodiments, the engineered phloem elements comprise synthetic tRNA-like structures. In some embodiments, engineered phloem mobility elements provided herein increase transport of an RNA cargo in a plant. In some embodiments, engineered phloem mobility elements provided herein increase transport of an RNA cargo to the meristem of the plant. In some embodiments, engineered phloem mobility elements provided herein transport an RNA cargo through the phloem and / or the xylem of a plant.

[0064] In some embodiments, the engineered phloem mobility element comprises between 70-85 nucleotides. In some embodiments, the engineered phloem mobility element comprises between 74-82 nucleotides. In some embodiments, the engineered phloem mobility element folds into the secondary structure with an energy partition function of less than -25 EpF. In some embodiments, the engineered phloem mobility element folds into the secondary structure 15MF-365821254Attorney Docket No.: 16536-20022.40with an energy partition function of less than -30 EpF. In some embodiments, the engineered phloem mobility element folds into the secondary structure with an energy partition function of less than -35 EpF. Energy partition functions of candidate engineered phloem mobility elements are shown in Tables 1-4.

[0065] Energy partition function (EpF) is a quantification of the free energy of the RNA secondary structure. EpF offers information regarding the thermodynamics of the RNA sequence. EpF calculations for RNA may be computed using the dynamic algorithm developed by McCaskill (1990), or variations on this algorithm that have been subsequently developed. Without being bound by theory, engineered phloem mobility elements comprising RNA sequences with EpF ranges indicated herein may be suitable for use due to the thermodynamic stability of their secondary structures.A. Secondary Structures of Phloem Mobility Elements

[0066] In some aspects, provided herein are engineered phloem mobility elements comprising a secondary structure selected from Structure I, Structure II, Structure III, and Structure IV, as defined herein. Structures are defined in dot-bracket notation, as shown in FIGs. 1A-1D. Representations of the three-dimensional structure of each secondary structure are shown in FIGs. 2A-2D.

[0067] In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:[Structure I]. In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:(((((((((((((•((((((•( )•)))))) )))))•((( )))•))))))))•• [Structure II], In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:[Structure III]. In some aspects, provided herein is an engineered phloem mobility element comprising an RNA having the secondary structure of:> [Structure IV], In some aspects, the secondary structure of the engineered phloem mobility element is maintained when the engineered phloem mobility element is linked to an RNA cargo. In some aspects, the engineered phloem mobility element comprises a sequence that is heterologous to the plant. In some embodiments, the engineered phloem mobility element has one of Structure I-IV and is not naturally- occurring.16MF-365821254Attorney Docket No.: 16536-20022.40

[0068] In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure I, and comprises a sequence selected from the group consisting of SEQ ID NOs: 1-595. In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure I, and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to any one of SEQ ID NOs: 1-595, wherein the sequence is not a naturally occurring sequence within the plant. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 370 and 593. In some embodiments, the engineered phloem mobility element comprises the sequence of SEQ ID NO: 370. In some embodiments, the engineered phloem mobility element comprises the sequence of SEQ ID NO: 593.

[0069] In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure II, and comprises a sequence selected from the group consisting of SEQ ID NOs: 596-655. In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure II, and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to any one of SEQ ID NOs: 596-655, wherein the sequence is not a naturally occurring sequence within the plant. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 648 and 653. In some embodiments, the engineered phloem mobility element comprises the sequence of SEQ ID NO: 648. In some embodiments, the engineered phloem mobility element comprises the sequence of SEQ ID NO: 653.

[0070] In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure III, and comprises a sequence selected from the group consisting of SEQ ID NOs: 656-690. In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure III, and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to any one of SEQ ID NOs: 656-690. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 688 and 689. In some embodiments, the engineered phloem mobility element comprises the sequence of SEQ ID NO: 688. In some embodiments, the engineered phloem mobility element comprises the sequence of SEQ ID NO: 689.

[0071] In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure IV, and comprises a sequence selected from the group 17MF-365821254Attorney Docket No.: 16536-20022.40consisting of SEQ ID NOs: 691-752. In some embodiments, the engineered phloem mobility element comprises a secondary structure of Structure IV, and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to any one of SEQ ID NOs: 691-752.

[0072] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising: a) a first stem of 9 nucleotides, b) a first stem loop structure comprising i) a second stem of five nucleotides comprising a bulge comprising one unpaired nucleotide and ii) a first loop comprising four nucleotides, c) a second stem loop structure comprising i) a third stem of five nucleotides and ii) a second loop comprising seven nucleotides, and d) a third stem loop structure comprising i) a third stem of four nucleotides and ii) a third loop comprising four nucleotides, further comprising: e) at least five unpaired nucleotides 5’ of the first stem, f) two unpaired nucleotides between the first stem and the first stem loop structure, g) one unpaired nucleotide between the second stem loop structure and the third stem loop structure, h) eight unpaired nucleotides between the third stem loop structure and the stem, and i) at least four unpaired nucleotides 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some embodiments, the engineered phloem mobility element provided herein comprises the secondary structure shown in FIG. 2A.

[0073] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising: a) a first stem of eight nucleotides, b) a first stem loop structure comprising i) a second stem of twelve nucleotides, comprising a first bulge comprising one unpaired nucleotide, a second bulge comprising one unpaired nucleotide, a third bulge comprising one unpaired nucleotide, and a first loop comprising six unpaired nucleotides, and ii) a second loop comprising seven nucleotides; and c) a second stem loop structure comprising i) a third stem of three nucleotides and ii) a third loop comprising seven nucleotides, further comprising: d) at least six unpaired nucleotides 5’ of the first stem, e) one unpaired nucleotide between the first stem loop structure and the second stem loop structure, f) one unpaired nucleotide between the second stem loop structure and the first stem, and g) at least two unpaired nucleotides 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some embodiments, the engineered phloem mobility element provided herein comprises the secondary structure shown in FIG. 2B.

[0074] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure 18MF-365821254Attorney Docket No.: 16536-20022.40comprising: a) a first stem of six nucleotides, b) a first stem loop structure comprising i) a second stem of four nucleotides, and ii) a first loop comprising nine nucleotides; c) a second stem loop structure comprising i) a third stem of five nucleotides, and ii) a second loop comprising seven nucleotides; and d) a third stem loop structure comprising i) a fourth stem of six nucleotides, and ii) a third loop comprising five nucleotides, further comprising: e) three unpaired nucleotides between the first stem and the first stem loop structure, f) one unpaired nucleotide between the first stem loop structure and the second stem loop structure, g) five unpaired nucleotides between the second stem loop structure and the third stem loop structure, h) one unpaired nucleotides between the third stem loop structure and the first stem, and i) at least one unpaired nucleotide 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some embodiments, the engineered phloem mobility element provided herein comprises the secondary structure shown in FIG. 2C.

[0075] In some aspects, provided herein is an engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising: a) a first stem of five nucleotides, comprising a first bulge comprising one unpaired nucleotide and a second bulge comprising one unpaired nucleotide; b) a first stem loop structure comprising i) a second stem of nine nucleotides, comprising a first bulge comprising three unpaired nucleotides, a second bulge comprising two unpaired nucleotides, a third bulge comprising two unpaired nucleotides, and a fourth bulge comprising three unpaired nucleotides, and ii) a first loop comprising four nucleotides; and c) a second stem loop structure comprising i) a third stem of eight nucleotides, comprising a first bulge comprising one unpaired nucleotide and a second bulge comprising one unpaired nucleotide, and ii) a second loop comprising four nucleotides, further comprising: d) three unpaired nucleotides between the first stem and the first stem loop structure, e) four unpaired nucleotides between the first stem loop structure and the second stem loop structure, f) one unpaired nucleotide between the second stem loop structure and the stem, and g) at least one unpaired nucleotide 3’ of the stem, wherein the RNA comprises a sequence that is not a naturally occurring sequence in the plant. In some embodiments, the engineered phloem mobility element provided herein comprises the secondary structure shown in FIG. 2D.

[0076] In some embodiments, the engineered phloem mobility element comprises between 70-85 nucleotides in length. In some embodiments, the engineered phloem mobility element comprises between 74-82 nucleotides. In some embodiments, the engineered phloem mobility element comprises a length of 82 nucleotides. In some embodiments, the engineered phloem19MF-365821254Attorney Docket No.: 16536-20022.40mobility element comprises a length of 78 nucleotides. In some embodiments, the engineered phloem mobility element comprises a length of 74 nucleotides.

[0077] In some embodiments, transport of the linked RNA cargo within the plant is increased compared to transport of the RNA cargo without the engineered phloem mobility element. In some embodiments, transport of the linked RNA cargo is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or greater. In some embodiments, transport of the linked RNA cargo is increased by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold within the plant compared to transport of the RNA cargo without the engineered phloem mobility element.

[0078] In some embodiments, the engineered phloem mobility element: a) comprises a secondary structure of Structure I, and comprises a sequence selected from the group consisting of SEQ ID NOs: 1-595; b) comprises a secondary structure of Structure II, and comprises a sequence selected from the group consisting of SEQ ID NOs: 596-655; c) comprises a secondary structure of Structure III, and comprises a sequence selected from the group consisting of SEQ ID NOs: 656-690; or d) comprises a secondary structure of Structure IV, and comprises a sequence selected from the group consisting of SEQ ID NOs: 691-752. In some embodiments, the engineered phloem mobility element comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to any one of SEQ ID NO: 1-752, wherein the sequence is not a naturally occurring sequence within the plant. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 370 and 593. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 648 and 653. In some embodiments, the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 688 and 689. In some embodiments, transport of the linked RNA cargo within the plant is increased compared to transport of the RNA cargo without the engineered phloem mobility element.

[0079] Secondary structure may be notated in the “dot-bracket notation” format (or “dotparenthesis notation”). Dot-bracket form consists of a balanced parentheses string containing a three-character alphabet selected from {.,(,)} that can be unambiguously converted into the corresponding RNA secondary structure. The characters code for an unpaired base an open base pair and a closed base pair ‘)’. This secondary structure notation can thus be used to represent the basic RNA secondary structures including hairpins, loops, internal loops, stems,20MF-365821254Attorney Docket No.: 16536-20022.40helixes, and bulges. For further discussion of dot-bracket notation, see, for example, Hofacker et al, 1994. Fast folding and comparison of RNA secondary structures. Monatschefte fur Chemie / Chemical Monthly, 125(2), which is hereby incorporated by reference in its entirety. Dot-bracket notation of the secondary structures of engineered phloem mobility elements disclosed herein are shown in FIGs.2A-2D. Structural diagrams of the secondary structures of engineered phloem mobility elements disclosed herein are shown in FIGs. 3A-3D.

[0080] RNA molecules comprise nucleotides, wherein a nucleotide may form a base pair with a complementary nucleotide to form a secondary structure. Base pairing in RNA canonically occurs between an adenine and a uracil, or between a guanine and a cytosine. Principles of nucleotide base pairing and sequence complementarity in nucleic acid molecules are well understood by those of skill in the art. In some cases, noncanonical base pairs may be formed (See, for example, Olsen et al, Effects of Noncanonical Base Pairing on RNA Folding: Structural Context and Spatial Arrangements of G-A Pairs, Biochemistry (2020), which is hereby incorporated by reference in its entirety). RNA may form complex and intricate basepairing interactions, which can be categorized into a variety of structural motifs including various types of helices, pseudoknots, and loops.B. RNA Cargo

[0081] In some embodiments, provided herein is an RNA molecule comprising the engineered phloem mobility element of any one of the preceding embodiments linked to the RNA cargo. In some embodiments, the engineered phloem mobility element is located 3’ to the RNA cargo. In some embodiments, the engineered phloem mobility element is located 5’ to the RNA cargo. In some embodiments, the RNA cargo is an mRNA molecule. In some embodiments, the RNA cargo encodes a Cas and / or comprises a guide RNA (gRNA). In some embodiments, the RNA cargo encodes a reporter, optionally a fluorescent reporter. In some embodiments, the reporter is a GFP or an eGFP variant. In some embodiments, provided herein is a nucleic acid encoding the RNA molecule of any one of the preceding embodiments. In some embodiments, provided herein is a vector comprising the nucleic acid of the preceding embodiment. In some embodiments, the RNA cargo is mRNA.

[0082] In some embodiments, the RNA cargo linked to the engineered phloem mobility element comprises multiple coding sequences. In some embodiments, the multiple coding sequences are linked via or separated by a self-cleaving peptide or an IRES sequence. In some embodiments, the RNA cargo comprises multiple cistrons. In some embodiments, the multiple cistrons are linked by a self-cleaving 2A peptide or an IRES sequence. In some embodiments,21MF-365821254Attorney Docket No.: 16536-20022.40the RNA cargo comprises a polycistronic construct. In some embodiments, the RNA cargo comprises two, three, four, or more coding sequences linked to an engineered phloem mobility element.

[0083] In some embodiments, provided herein is an RNA molecule comprising an engineered phloem mobility element as described herein, linked to the RNA cargo. In some embodiments, the engineered phloem mobility element is located 3’ to the RNA cargo. In some embodiments, the engineered phloem mobility element is located 5’ to the RNA cargo. In some embodiments, the engineered phloem mobility element is located 3’ to a first RNA cargo and 5’ to a second RNA cargo. In some embodiments, provided herein is an RNA molecule comprising an RNA cargo linked to at least one engineered phloem mobility element described herein.

[0084] In some embodiments, the RNA cargo comprises nucleic acid encoding a Cas nuclease and one or more gRNAs fused 5’ to the Cas. In some embodiments, the RNA cargo additionally comprises a polyA tail. In some embodiments, the RNA cargo does not comprise a polyA tail. In some embodiments, the one or more gRNAs fused 5’ to the Cas are processed by the translated nuclease (see, for example, WO2021081200, which is hereby incorporated by reference in its entirety). In some embodiments, the Cas is a Cas 12s or Casl2i.

[0085] In some embodiments, the RNA cargo comprises a retron. Retrons are bacterial retroelements that comprise a cassette encoding a reverse transcriptase and a non-coding RNA (ncRNA), and produce noncoding intracellular DNAs including multicopy single- stranded DNA (msDNA) from the reverse transcription reaction. In some cases, a retron element may also comprise one or more genes encoding an effector protein. Retron systems may be harnessed for genome editing purposes (see, for example, Simon AJ et al, Retrons and their applications in genome engineering. Nucleic Acids Res (2019) Oct 10;47(21)l 1007-11019). In some embodiments, a retron system may be used in combination with the engineered phloem mobility elements as described herein. In some embodiments, a retron may be provided to a meristem cell of a plant, wherein a Cas and one or more guide RNAs linked to an engineered phloem mobility element are separately introduced to the meristem cell, wherein the retron provides a donor DNA template that is introduced into the genome of the meristem cell following the cleavage of double-stranded DNA in the meristem cell by the Cas.

[0086] In some embodiments, the RNA cargo comprises an mRNA molecule. In some embodiments, the mRNA molecule encodes a reporter, detectable label, or fluorophore, for example, a fluorophore such as eGFP or mCherry. Any fluorescent reporter, such as but not limited to a green, red, blue, far-red, or yellow fluorescent reporter, may be suitable for use. Particular examples of labels that may be used in accordance with the provided embodiments 22MF-365821254Attorney Docket No.: 16536-20022.40comprise, but are not limited to phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenical acetyl transferase, and urease. In other embodiments, a reporter tag such as an HA tag may be included. Such reporter mRNAs may be useful for evaluating efficiency of the construct, for identifying progeny, and for other uses. In some embodiments, mRNA molecules encoding reporters may be included in combination with other RNA cargos.

[0087] In some embodiments, the RNA cargo comprises a siRNA molecule, wherein the RNA cargo is linked to an engineered phloem mobility element. In some embodiments, the RNA cargo comprises a dsRNA, wherein the RNA cargo is linked to an engineered phloem mobility element. In some embodiments, the RNA cargo comprises a ssRNA, wherein the RNA cargo is linked to an engineered phloem mobility element. In some embodiments, the RNA cargo is provided to a plant.

[0088] In some embodiments, provided herein is a nucleic acid encoding an RNA molecule comprising an engineered phloem mobility element as described herein linked to an RNA cargo. In some embodiments, provided herein is a vector comprising the nucleic acid encoding an RNA molecule comprising an engineered phloem mobility element as described herein linked to an RNA cargo. In some embodiments, the vector is an engineered T-DNA vector. In some embodiments, the vector is provided to the plant via a hairy root system.

[0089] In some embodiments, the RNA cargo further comprises a sequence comprising or encoding a cassette for selection. In some embodiments, the cassette encodes a resistance gene against a pesticide, bacteria, virus, or other agent. In some embodiments, the cassette is a glyphosate resistance cassette.i. Cas nucleases and guide RNAs

[0090] In some embodiments, the RNA cargo comprises nucleic acid encoding a Cas nuclease. In some embodiments, a nucleic acid encoding a gRNA for the Cas nuclease is provided separately to the plant. In some embodiments, the nucleic acid encoding the gRNA for the Cas nuclease is linked to a second phloem mobility element. In some embodiments, the second phloem mobility element is an engineered phloem mobility element of any of the preceding embodiments. In some embodiments, the second phloem mobility element 23MF-365821254Attorney Docket No.: 16536-20022.40comprises or is derived from: i. a Flower Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or ii. an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop. In some embodiments, the RNA cargo comprises nucleic acid encoding a gRNA for a Cas nuclease. In some embodiments, the Cas nuclease is constitutively expressed in the plant or in the roots of the plant.

[0091] In some embodiments, the plant comprises a rootstock and a scion grafted onto the rootstock. In some embodiments, nucleic acid encoding the Cas nuclease is expressed in the rootstock, wherein the nucleic acid encoding the Cas nuclease is linked to a second phloem mobility element. In some embodiments, the second phloem mobility element is an engineered phloem mobility element of any one of the preceding embodiments. In some embodiments, the second phloem mobility element comprises or is derived from: i. a Flower Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); or ii. an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.

[0092] In some embodiments, the RNA cargo linked to the engineered phloem mobility element comprises i) nucleic acid encoding a Cas nuclease and ii) a gRNA for the Cas nuclease. In some embodiments, the nucleic acid encoding the Cas nuclease is transported to the meristem, wherein the Cas nuclease is translated in the meristem. In some embodiments, a genomic target within a cell in the meristem is edited. In some embodiments, transport of the nucleic acid encoding the Cas nuclease is increased when the Cas nuclease is linked to the engineered phloem mobility element compared to transport of a Cas nuclease that is not linked to an engineered phloem mobility element. In some embodiments, increased editing of a genomic target within the meristem is increased when the RNA cargo comprising the Cas nuclease is linked to an engineered phloem mobility element compared to an RNA cargo comprising a Cas nuclease not linked to an engineered phloem mobility element.

[0093] In some embodiments, the plant comprises a rootstock and a scion grafted onto the rootstock. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and / or rootstock is a dicot. In some embodiments, the plant is a dicot. In some embodiments, the scion and / or rootstock is a monocot. In some embodiments, the plant is a monocot. In some embodiments, the rootstock and / or scion, or plant is soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana, or wheat. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in dicots, optionally wherein the 24MF-365821254Attorney Docket No.: 16536-20022.40nucleic acid encoding the Cas nuclease is codon-optimized for expression in soybean. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in monocots. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in corn, soy, or wheat. In some embodiments, the method further comprises retrieving a progeny of the plant, wherein the progeny has an altered genome.

[0094] In some embodiments, the guide RNA further comprises: (a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide RNA; or (b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide RNA; or (c) both (a) and (b); wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar. In some embodiments, each of the one or more modified nucleotides is independently selected from the group consisting of 2’-O-methyl nucleotide, a 2’-0-methyl-3’phosphorothioate nucleotide, a 2’-O-methyl-3’phosphonoacetate nucleotide, and a 2’-0-methyl-3’-phosphonothioacetate nucleotide. In some embodiments, the one or more modified nucleotides comprises a modified internucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group. In some embodiments, the gRNA comprises a sequence that is heterologous to the plant.

[0095] In some embodiments, the RNA cargo encodes a transposon-associated RNA-guided nuclease. In some embodiments, the transposon-associated RNA-guided nuclease is a TnpB nuclease (see, for example, US20240084332, which is hereby incorporated by reference in its entirety). In some embodiments, the RNA cargo additionally comprises one or more RNA guides for the TnpB nuclease. In some embodiments, the one or more RNA guides comprise one or more coRNA molecules. In some embodiments, the one or more RNA guides are located 5’ to the TnpB nuclease. In some embodiments, the transposon-associated RNA-guided nuclease is an IscB nuclease (see, for example, US20230392131, which is hereby incorporated by reference in its entirety). In some embodiments, the RNA cargo additionally comprises one or more RNA guides for the IscB nuclease. In some embodiments, the one or more RNA guides comprise one or more coRNA molecules. In some embodiments, the one or more RNA guides are located 5’ to the IscB nuclease.25MF-365821254Attorney Docket No.: 16536-20022.40C. Linkers

[0096] In some aspects, engineered phloem mobility elements described herein are linked to RNA cargo, and facilitate or increase transport of said RNA cargo within a plant. Engineered phloem mobility elements may be linked to RNA cargo by any appropriate linker. Linkers suitable for use with RNA molecules may comprise one or more nucleotides and are known to those of skill in the art. In some embodiments, the presence of a linker may allow for expression of multiple RNA cargo cistrons. In some embodiments, an RNA molecule comprising an RNA cargo and an engineered phloem mobility element described herein additionally comprises a linker. In some embodiments, the linker comprises one or more nucleotides. In some embodiments, the linker is a glycine linker. In some embodiments, engineered phloem mobility elements described herein are directly linked to RNA cargo in an RNA delivery molecule, wherein the RNA delivery molecule does not comprise a linker.

[0097] In some embodiments, an RNA cargo linked to an engineered phloem mobility element is flanked by one or more processing elements, so that functional RNAs are excised inside the cells. Exemplary processing elements include hammerhead ribozymes, Csy4, and tRNAs (see Mikami et al., Plant Cell Physiol. 2017, 58(11): 1857-1867; and US Patent No.10,308,947). Ribozymes can autocatalytically cleave the RNA molecule comprising the RNA cargo and the engineered phloem mobility element to release the RNA cargo from a polycistronic transcript and / or remove additional 5’ or 3’ sequence(s) around the RNA cargo. tRNAs are processed by elements of the cell’s endogenous tRNA system, such as RNase P, RNase Z, and RNase E, and tRNA sequences or pre-tRNA sequences can also be used to release an RNA cargo flanked by processing elements from a polycistronic transcript and / or remove additional 5’ or 3’ sequence(s) around the RNA cargo.

[0098] In some embodiments, an RNA cargo linked to an engineered phloem mobility element is flanked by one or more direct repeats (DR) of a CRISPR array. In some embodiments, the direct repeats are suitable for processing by a Casl2a or Casl2i without the need of a tracrRNA.III. Methods of Transporting RNA Cargo to the Meristem of a Plant

[0099] The present application provides methods of transporting an RNA cargo to the meristem of a plant (i.e., to a meristem cell of the plant), wherein the method comprises providing to the plant the RNA cargo linked to an engineered phloem mobility element described herein (i.e., described in section II).26MF-365821254Attorney Docket No.: 16536-20022.40

[0100] In some aspects, provided herein is a method of transporting an RNA cargo to a meristem cell of a plant comprising providing to the plant the RNA cargo linked to the engineered phloem mobility element of any of the preceding embodiments. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the plant via a T-DNA vector. In some embodiments, the T-DNA vector is delivered to a root of the plant. In some embodiments, the T-DNA vector comprises a Ri plasmid from Agrobacterium rhizogenes. In some embodiments, the RNA cargo linked to the phloem mobility element is delivered to the root of the plant by an Agrobacterium rhizogenes transformation. In some embodiments, the Agrobacterium rhizogenes transformation produces transgenic hairy roots.

[0101] In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to a root of the plant by injecting a composition comprising the RNA cargo linked to the engineered phloem mobility element into the root. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the plant root by incubating the root with a composition comprising the RNA cargo linked to the engineered phloem mobility element. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the plant root by an Agrobacterium rhizogenes transformation. In some embodiments, the RNA cargo is transported by the plant vascular system when linked to the engineered phloem mobility element. In some embodiments, the RNA cargo is transported through the xylem or the phloem when linked to the engineered phloem mobility element.A. Editing of a plant with genome editing reagents linked to engineered phloem mobility elements.

[0102] The present application provides methods of delivering RNA cargo comprising one or more genome editing reagents linked to an engineered phloem mobility element to a plant, wherein the one or more genome editing reagents are delivered to the meristem of the plant, and thereby editing a genomic target in a meristem cell of the plant. In some embodiments, a method provided herein comprises delivering an RNA cargo comprising a guide RNA (gRNA) directed to the genomic target in the meristem cell in the plant by; and delivering a Cas nuclease to the plant, wherein the Cas nuclease and / or the gRNA are linked to a phloem mobility element when delivered to the plant, wherein the Cas nuclease and the guide RNA are transported to27MF-365821254Attorney Docket No.: 16536-20022.40the meristem of the plant, and wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the plant. In some embodiments, the guide RNA and / or the Cas nuclease are provided to the plant via Agrobacterium-mediated delivery. In some embodiments, the guide RNA and / or the Cas nuclease are provided via virus-mediated delivery.

[0103] In some embodiments, the genome editing reagents are provided to the plant by infection with an Agrobacterium rhizogenes vector. In some embodiments, the Agrobacterium rhizogenes vector carries at least one guide RNA, wherein the at least one guide RNA is linked to an engineered phloem mobility element. In some embodiments, the Agrobacterium vector is linked to at least one guide RNA, wherein the at least one guide RNA is linked to an engineered phloem mobility element. In some embodiments, the Cas enzyme is delivered to the plant in the same Agrobacterium vector carrying the gRNA. In some embodiments, the Cas enzyme is delivered to the soybean plant by a second vector comprising nucleic acid encoding the Cas enzyme. In some embodiments, the second vector is an Agrobacterium rhizogenes vector. In some embodiments, the second vector is a BMPV vector. In some embodiments, the method further comprises infecting the plant with a plurality of vectors, each vector comprising a gRNA and / or the Cas enzyme, wherein the gRNA and / or the Cas enzyme are linked to an engineered phloem mobility element. In some embodiments, the plurality of vectors co-infect the plant simultaneously or infect the plant in more than one round of infection. In some embodiments, the vector also includes nucleic acid encoding Casl2f nuclease. In some embodiments, nucleic acid encoding Casl2f nuclease and the guide RNA are provided in the same vector, wherein the nucleic acid encoding Casl2f nuclease and the guide RNA are linked to an engineered phloem mobility element. In some embodiments, the nucleic acid encoding Casl2f nuclease and the guide RNA are provided in different vectors, wherein the nucleic acid encoding Casl2f nuclease and / or the guide RNA are each linked to an engineered phloem mobility element. In some embodiments, nucleic acid encoding the Cas nuclease and the guide RNA are provided in the same vector. In some embodiments, the nucleic acid encoding the Cas nuclease and the guide RNA are provided in different vectors.

[0104] In some embodiments, the plant expresses a Cas nuclease prior to transformation with the T-DNA vector, wherein the Cas nuclease is linked to an engineered phloem mobility element when expressed in the plant. In some embodiments, the plant is a Cas editor line. In some embodiments, the plant has a Cas enzyme stably integrated into its genome. In some embodiments, the genome editing reagents are overexpressed in the plant. In some embodiments, the plant overexpresses the Cas enzyme. In some embodiments, the plant 28MF-365821254Attorney Docket No.: 16536-20022.40overexpresses Cas9. In some embodiments, the expression of the nuclease is under control of an inducible promoter. The plant overexpressing a Cas nuclease may be generated through transformation techniques. In some embodiments, the plant overexpresses Casl2, such as Casl2f„ Casl2i, or Casl2a. In some embodiments, the plant is engineered to express a Cas nuclease prior to viral vector delivery. Infecting a plant expressing a Cas nuclease may be advantageous, as this method avoids the need to deliver the Cas nuclease via highly limited viral vector cargo space. In some embodiments, the plant overexpresses the Cas in a meristem cell. In some embodiments, the plant overexpresses the Cas in cells other than the meristem cell and the Cas is transported to the meristem cell via an engineered phloem mobility element. In some embodiments, an RNA cargo comprising a guide RNA is provided to the plant, wherein the RNA cargo is linked to an engineered phloem mobility element. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is transported to the meristem cell. In some embodiments, the guide RNA and the Cas nuclease edit the meristem cell.

[0105] In some embodiments, the present application provides methods of providing an RNA cargo comprising a guide RNA linked to an engineered phloem mobility element to a plant, wherein the plant expresses a Cas nuclease. In some embodiments, the RNA cargo comprises one or more guide RNAs. In some embodiments, the plant comprises a rootstock, wherein the rootstock expresses Cas. In some embodiments, a meristem cell of the plant expresses Cas. In some embodiments, the RNA cargo is delivered to the plant via the roots via Agrobacterium-mediated transformation, wherein the RNA cargo is transported to the meristem cell of the plant. In some embodiments, the guide RNA and the Cas edit the meristem cell.

[0106] The present application also provides methods of providing RNA cargo comprising genome editing reagents linked to an engineered phloem mobility element to a plant scion and / or rootstock, and thereby editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a Cas nuclease and nucleic acid encoding a guide RNA for the Cas nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas nuclease are linked to a phloem mobility element. A rootstock provides nucleic acid encoding an engineered phloem mobility element linked to genome editing reagents, i.e., a Cas nuclease and a guide RNA for the Cas nuclease, to the plant vascular system. In some embodiments, an engineered phloem mobility element linked to RNA encoding the Cas9 nuclease or Cas 12 nuclease and the guide RNA are transported from the rootstock to the scion by the plant vascular system. In some embodiments, an engineered phloem mobility element linked to RNA encoding the Cas9 nuclease or Casl229MF-365821254Attorney Docket No.: 16536-20022.40nuclease and the guide RNA are transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding the Cas9 nuclease or Casl2 nuclease is translated in the scion. In some embodiments, a meristem cell of the scion is edited.

[0107] In some embodiments, provided herein is a rootstock comprising nucleic acid encoding a Cas9 nuclease or Casl2 nuclease and nucleic acid encoding a guide RNA for the Cas9 nuclease or Casl2 nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease are linked to an engineered phloem mobility element. In some embodiments, a guide RNA (gRNA) for the Cas9 nickase or Casl2 nuclease is also linked to the engineered phloem mobility element. In some embodiments, the guide RNA and the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease are linked to the engineered phloem mobility element in separate vectors or constructs. In some embodiments, the guide RNA and the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease are linked to the engineered phloem mobility element in the same construct, optionally a polycistronic construct.

[0108] In some embodiments, the genome editing reagents linked to the engineered phloem mobility element are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots. In some embodiments, the Cas enzyme linked to the engineered phloem mobility element is provided to the soybean plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a soybean plant with transgenic hairy roots. In some embodiments, the nucleic acid encoding the Cas9 nuclease or Cas 12 nuclease and the nucleic acid encoding the guide RNA are provided in the same vector and are linked to an engineered phloem mobility element. In some embodiments, the nucleic acid encoding the Cas9 nuclease or Cas 12 nuclease and the nucleic acid encoding the guide RNA (gRNA) are provided in different vectors, wherein one or both are linked to an engineered phloem mobility element. In some embodiments, the vector is a viral vector. In some embodiments, virus-mediated delivery comprises using a viral vector comprising the gRNA. In some embodiments, the vector is a T-DNA vector. In some embodiments, the vector is a viral vector or a T-DNA vector.

[0109] In some embodiments, an RNA cargo linked to an engineered phloem mobility element provided herein is provided to the plant via virus-mediated delivery, wherein the virus-mediated delivery comprises infecting the plant with an inoculum comprising the viral vector. The linking of the engineered phloem mobility element to the gRNA and / or the nucleic acid encoding the Cas nuclease results in the genome editing reagent(s) being transported through the phloem or xylem, and / or transported to cells of the meristem of the plant. Modifications or 30MF-365821254Attorney Docket No.: 16536-20022.40edits made in the plant meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. Modifications or edits made in the plant meristem are heritable as the meristem cells will form the reproductive tissues of the plant, including the gametes.

[0110] In some embodiments, virus-mediated delivery comprises infecting the plant with an inoculum comprising the viral vector. The engineered phloem mobility element linked to the gRNA and / or the nucleic acid encoding the Cas nuclease results in the genome editing reagent(s) being transported to cells of the meristem of the plant through viral infection of the plant roots via Agrobacterium-mediated transformation and subsequent transport to the meristem of the plant. Modifications or edits made in the plant meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. Modifications or edits made in the plant meristem are heritable as the meristem cells will form the reproductive tissues of the plant, including the gametes.

[0111] The present application provides methods of editing a genomic target in a meristem cell of a plant comprising delivering a guide RNA (gRNA) to the root of the plant that is directed to the genomic target in the meristem cell; and delivering a Cas nuclease to the root of the plant via a hairy root system, wherein the gRNA and / or the Cas nuclease is linked to an engineered phloem mobility element and are delivered to the meristem of the plant, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the plant. In some embodiments, virus-mediated delivery comprises using a T-DNA vector comprising the gRNA.

[0112] In some embodiments, the genomic target is in a scion. In some embodiments, the Cas nuclease is delivered to the scion by transport from a grafted rootstock. Accordingly, the present application also provides, in some embodiments, methods of editing a genomic target in a scion comprising grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease linked to an engineered phloem mobility element; and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery, optionally wherein the scion comprises a leaf, a shoot, a stem, or other vegetative tissue. The rootstock provides a Cas nuclease to the scion, transported through the grafting site due to the engineered phloem mobility element. Any guide RNA can then be delivered to the scion by means of viral delivery, such mean including but not limited to direct leaf rub inoculation with infectious sap, inoculation with an infectious cDNA plasmid, and transformation of the root with a bacterium comprising a vector comprising a recombinant plant virus.. As plant viruses are designed to deliver genetic cargo to plant cells, utilization of 31MF-365821254Attorney Docket No.: 16536-20022.40such methods to deliver genomic editing reagents is very effective. A scion may be edited at a given locus by such methods without the need for a transgene insertion (i.e. the genome editing reagents) in the genome of the scion. By other methods, integration of edits or desired traits into elite germplasm would take many crosses and require additional time and resources.B. Providing RNA Cargo to a grafted scion via rootstock

[0113] The present application provides methods of providing RNA cargo comprising genome editing reagents linked to an engineered phloem mobility element to a plant rootstock grafted to a plant scion, and thereby editing a genomic target in the plant scion. In some embodiments, a method provided herein comprises grafting the scion onto a rootstock comprising nucleic acid encoding a Cas nuclease and nucleic acid encoding a guide RNA for the Cas nuclease, wherein the nucleic acid encoding the guide RNA and / or the nucleic acid encoding the Cas nuclease are linked to an engineered phloem mobility element. A rootstock provides nucleic acid encoding genome editing reagents, i.e., a Cas nuclease and a guide RNA for the Cas nuclease, linked to an engineered phloem mobility element, to the plant vascular system. In some embodiments, RNA encoding the Cas nickase or nuclease and the guide RNA linked to an engineered phloem mobility element are transported from the rootstock to the scion by the plant vascular system. In some embodiments, RNA encoding the Cas nickase or nuclease and the guide RNA linked to an engineered phloem mobility element are transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding the Cas nickase or nuclease is translated in the scion. In some embodiments, a meristem of the scion is edited.

[0114] In some embodiments, the RNA cargo comprises RNA encoding a Cas nuclease. In some embodiments, the Cas nuclease is a Cas 12 nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease. In some embodiments, the Cas nuclease is any Cas nuclease suitable for use in plants. In some embodiments, RNA encoding the Cas9 nuclease or Cas 12 nuclease and the guide RNA linked to an engineered phloem mobility element are transported from the rootstock to the scion by the plant vascular system. In some embodiments, RNA encoding the Cas9 nuclease or Cas 12 nuclease and the guide RNA linked to an engineered phloem mobility element are transported from the rootstock to the scion through the phloem. In some embodiments, RNA encoding the Cas9 nuclease or Cas 12 nuclease is translated in the scion. In some embodiments, a meristem of the scion is edited.32MF-365821254Attorney Docket No.: 16536-20022.40

[0115] In some embodiments, provided herein is a rootstock comprising nucleic acid encoding a Cas9 nuclease or Casl2 nuclease and nucleic acid encoding a guide RNA for the Cas9 nuclease or Casl2 nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease are linked to an engineered phloem mobility element described herein.

[0116] In some embodiments, the genome editing reagents are provided to the rootstock by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a rootstock with transgenic hairy roots. In some embodiments, the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease and the nucleic acid encoding the guide RNA are provided in the same vector, and are each linked to an engineered phloem mobility element as described herein. In some embodiments, the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease and the nucleic acid encoding the guide RNA are provided in different vectors, wherein the Cas9 nuclease or Casl2 nuclease and the nucleic acid encoding the guide RNA are each linked to an engineered phloem mobility element as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a T-DNA vector. In some embodiments, the vector is a viral vector or a T-DNA vector.

[0117] In some embodiments, the rootstock comprises nucleic acid encoding two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the nucleic acid encoding each of the two or more, three or more, four or more, or five or more guide RNAs is linked to an engineered phloem mobility element.

[0118] In some embodiments, the rootstock further comprises nucleic acid encoding a detectable marker linked to a nucleic acid encoding or comprising an engineered phloem mobility element.

[0119] In some embodiments, a scion is grafted onto the rootstock. The engineered phloem mobility element linked to the nucleic acid encoding the genome editing reagents results in the genome editing reagents being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction. Nucleic acid encoding the genome editing reagents are translated in the cytosol of cells of the scion meristem and imported into meristem nuclei, whereupon the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. Nucleic acid encoding the genome editing reagents are translated in the cytosol of cells of the scion meristem and imported into meristem cells, whereupon the genome of the meristem cell is edited. Edits made in the scion33MF-365821254Attorney Docket No.: 16536-20022.40meristem cell are heritable as the meristem cell will form the reproductive tissues of the plant, including the gametes.

[0120] In some embodiments, by this method, editing of the scion meristem can be accomplished without the introduction of a transgene to the genome of the scion. The scion and resulting progeny will be genetically edited without containing sequences encoding the Cas nuclease and the guide RNA in its genome. This will result in more consistent editing results, as there will be no element of randomness as to where a transgene will insert itself in the genome, or what levels of expression will result from each randomized insertion locus. The provided methods will also result in faster breeding and safety programs, as there is no possibility of off-target effects from insertion of a transgene into an inopportune location in the genome, and there is no need for additional breeding or selection to remove a transgene encoding genome editing reagents from the scion genome. Additionally, the provided line of rootstocks comprising genome editing reagents can be a modular tool for editing a number of existing elite plant lines. A single rootstock line can be used to transform many grafted scions, without the need to transform each scion. The provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.C. Delivery of guide RNA cargo for editing a plant

[0121] In some embodiments, the present application provides methods of editing a genomic target in a plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding a Cas nuclease is linked to an engineered phloem mobility element, and delivering to the scion a guide RNA for the Cas nuclease, optionally wherein the guide RNA for the Cas nuclease is also linked to an engineered phloem mobility element. The present application also provides methods of editing a genomic target in a meristem cell of a plant comprising delivering a guide RNA (gRNA) directed to the genomic target in the meristem cell in the plant by a hairy root system, wherein the gRNA is linked to an engineered phloem mobility element described herein; and delivering a Cas nuclease to the plant, wherein the Cas nuclease is linked to an engineered phloem mobility element described herein, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the plant, thereby editing the genomic target in the meristem cell. In some embodiments, the Cas nuclease linked to an engineered phloem mobility element described herein is delivered to the plant by transformation methods. In some embodiments,34MF-365821254Attorney Docket No.: 16536-20022.40the Cas enzyme linked to an engineered phloem mobility element described herein is delivered to the plant by infection with Agrobacterium. In some embodiments, the infection with Agrobacterium comprises infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. A rootstock provides nucleic acid encoding a Cas nuclease to the plant vascular system. In some embodiments, a scion is grafted onto the rootstock. In some embodiments, delivery via a hairy root system comprises using a T-RNA vector comprising the gRNA linked to an engineered phloem mobility element described herein.

[0122] In some embodiments, the Cas nuclease is linked to an engineered phloem mobility element. The engineered phloem mobility element linked to nucleic acid encoding the Cas nuclease results in the nucleic acid encoding the Cas nuclease being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction. Nucleic acid encoding the Cas nuclease is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei. In some embodiments, RNA encoding the Cas nuclease is translated in the meristem. In some embodiments, the Cas nuclease is delivered by transport from another part of the plant through the plant vascular system.

[0123] In some embodiments, the method comprises delivering two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each linked to an engineered phloem mobility element. In some embodiments, two or more guide RNAs are encoded by a single precursor RNA. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. In some embodiments, the viral vector carries at least two guide RNAs, at least three guide RNAs, at least four guide RNAs, at least five guide RNAs, at least six guide RNAs, at least seven guide RNAs, or at least eight guide RNAs. In some embodiments, the method comprises delivering a viral vector carrying two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the viral vector comprises at least two gRNAs, at least three gRNAs, at least four gRNAs, at least five gRNAs, at least six gRNAs, at least seven gRNAs, or at least eight gRNAs, optionally wherein each gRNA is directed to a different genomic target in the plant or a same genomic target in the plant. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each joined to the vector. In some embodiments, each guide RNA is directed to a different gene of interest in the plant. In some embodiments, each guide RNA is directed to the same gene of interest. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. In some 35MF-365821254Attorney Docket No.: 16536-20022.40embodiments, the vector carries at least two guide RNAs, at least three guide RNAs, at least four guide RNAs, at least five guide RNAs, at least six guide RNAs, at least seven guide RNAs, or at least eight guide RNAs. In some embodiments, the method comprises delivering a vector carrying two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each joined to the vector.

[0124] A guide RNA linked to an engineered phloem mobility element may be delivered to the meristem in a variety of ways. A guide RNA may be delivered to the meristem cell in a variety of ways. For example, in some embodiments, the guide RNA is delivered to the scion or directly to the meristem of the scion. In some embodiments, the guide RNA is delivered to the rootstock and transported into the scion. In some embodiments, the guide RNA is produced in vitro. In some embodiments, the guide RNA is methylated in vitro, such as by an RNA methylase, to promote mobility. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. Delivery of the guide RNA can occur through the following non-exhaustive list: through use of an RNA spray comprising the guide RNA and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by application of a composition comprising the guide RNA onto a leaf after rubbing the leaf with 200 grit sandpaper with a dowel; by spraying onto a leaf very fine glass beads coated with a composition comprising the guide RNA; by injection of a composition comprising the guide RNA into the stem; by infiltration of the leaf with a composition comprising the guide RNA; by direct uptake in the roots of a composition comprising the guide RNA; or by biolistic delivery to leaves or other tissue with circular DNA expressing the guide RNA. In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and / or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA. In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and / or meristem. In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem. In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needleless syringe or vacuum pump. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the nuclease inhibitor comprises an RNase inhibitor. In some embodiments, delivery of the guide comprises biolistic 36MF-365821254Attorney Docket No.: 16536-20022.40transformation of nucleic acid encoding the guide RNA into the leaf, shoot, shoot, stem, and / or meristem. In some embodiments, the vector is delivered to a leaf, shoot, stem, root, or other vegetative tissue. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA.

[0125] Delivery of a vector carrying the guide RNA can occur, for example, by application of a composition comprising the vector carrying the guide RNA onto a leaf after rubbing the leaf with 200 grit sandpaper with a dowel, by injection of a composition comprising the vector carrying the guide RNA into the stem, or by infiltration of the leaf with a composition comprising the vector carrying the guide RNA. In some embodiments, infecting the plant comprises applying an inoculum comprising the vector carrying the gRNA. In some embodiments, infecting the plant comprises inoculating the plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the vector carrying the gRNA.

[0126] In some embodiments, virus-mediated delivery comprises infecting the plant with an inoculum comprising the vector. In some embodiments, infecting the plant comprises applying an inoculum comprising a viral vector or a T-DNA vector carrying the gRNA. In some embodiments, infecting the plant comprises inoculating the plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector or the T-DNA vector carrying the gRNA.

[0127] In some embodiments, the provided method of editing a genomic target in a scion comprises grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease linked to an engineered phloem mobility element; and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery. In some embodiments, the method further comprises transforming the rootstock with nucleic acid encoding the Cas nuclease, wherein the nucleic acid encoding the Cas nuclease is linked to the engineered phloem mobility element, prior to grafting. In some embodiments, the scion comprises a leaf, a shoot, a stem, and / or a meristem. In other aspects, provided herein are methods of editing a genomic target in the meristem cell of a plant comprising transforming the root of the plant with nucleic acid encoding a Cas nuclease; and delivering a guide RNA for the Cas nuclease to a leaf, a shoot, a stem, and / or meristem of the plant by virus-mediated delivery, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. In some embodiments, delivery of the guide RNA comprises inoculating the leaves, shoot, stem, and / or meristem with a composition comprising a recombinant plant virus that comprises the guide RNA or a polynucleotide encoding the guide RNA, wherein the guide 37MF-365821254Attorney Docket No.: 16536-20022.40RNA or polynucleotide is linked to an engineered phloem mobility element. In some embodiments, delivery of the guide RNA linked to the engineered phloem mobility element comprises transforming the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, delivery of the guide RNA linked to the engineered phloem mobility element comprises transforming the leaves, shoot, stem, and / or meristem of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, infecting the plant comprises inoculating the plant’s leaves, shoot, stem, roots, or other vegetative tissue with a composition comprising the viral vector that contains the gRNA. In some embodiments, delivery of the guide RNA comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus. In some embodiments, the bacterium further comprises a binary vector comprising the Cas nuclease. In some embodiments, the virus-mediated delivery of the methods disclosed herein comprises transforming the root of the plant with a bacterium comprising a binary vector comprising a recombinant plant virus comprising the guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA or the nucleic acid encoding the guide RNA are linked to an engineered phloem mobility element, optionally wherein the bacterium further comprises a binary vector comprising a nucleic acid encoding the Cas nuclease.

[0128] In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA linked to the engineered phloem mobility element is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA linked to the engineered phloem mobility element is transported to the scion through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA linked to the engineered phloem mobility element is transported to the meristem. In some embodiments, RNA encoding the Cas nuclease linked to the engineered phloem mobility element is translated in the meristem. In some embodiments, one or more meristematic cells is edited or modified.

[0129] In some embodiments, the guide RNA linked to the engineered phloem mobility element is transported to the meristem of the plant scion, or is provided to the meristem of the plant scion directly. The guide RNA linked to the engineered phloem mobility element is imported into the meristem nuclei. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease into the meristem nuclei, the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes.38MF-365821254Attorney Docket No.: 16536-20022.40

[0130] The provided methods allow for fast and modular editing of a multitude of plants without the introduction of a transgene to the genome of the edited plant and / or edited scion. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on rootstock plants providing the Cas nuclease, and different guide RNAs linked to the engineered phloem mobility elements can be delivered to the different plant scions. Because there isn’t a different transgene being inserted into a different location in each plant scion, this allows for direct comparison of the results of providing different guide RNAs, including but not limited to comparison of efficiency of method of delivery, editing efficiency of different guide RNAs, and phenotypic changes as a result of edits induced by different guide RNAs. The provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.

[0131] Additionally, the provided methods allow for a reduced number of required transformation events. The rootstock providing the Cas nuclease can be used with a wide variety of delivered guide RNAs, increasing the modularity of the editing system. In some embodiments, the Cas nuclease is linked to a phloem mobility element.1). Delivery of guide RNA cargo to edit a scion

[0132] The present application provides methods of delivering an RNA cargo comprising a guide RNA and linked to an engineered phloem mobility element to a plant scion, and thereby editing a genomic target in the plant scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding a Cas nuclease is linked to an engineered phloem mobility element, and delivering to the scion a guide RNA linked to an engineered phloem mobility element for the Cas nuclease. In some embodiments, the Cas nuclease linked to the engineered phloem mobility element is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. A rootstock provides nucleic acid encoding a Cas nuclease linked to an engineered phloem mobility element to the plant vascular system. In some embodiments, a scion is grafted onto the rootstock. The engineered phloem mobility element to nucleic acid encoding the Cas nuclease results in the nucleic acid encoding the Cas nuclease being transported to cells of the meristem of the scion through the plant vascular system, which connects the rootstock to the scion through the graft junction. Nucleic acid encoding the Cas nuclease linked to the engineered phloem mobility element is translated39MF-365821254Attorney Docket No.: 16536-20022.40in the cytosol of cells of the scion meristem and imported into meristem nuclei. In some embodiments, the guide RNA is linked to an engineered phloem mobility element when delivered to the scion.

[0133] In some embodiments, the method comprises delivering two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each linked to an engineered phloem mobility element. In some embodiments, two or more guide RNAs are encoded by a single precursor RNA. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat.

[0134] In some embodiments, an RNA cargo may be a guide RNA linked to an engineered phloem mobility element. In some embodiments, the RNA cargo may be delivered to the plant, and subsequently transported within the plant to the meristem of the plant, in a variety of ways. For example, in some embodiments, the RNA cargo comprising the guide RNA linked to the engineered phloem mobility element is delivered to the scion or directly to the meristem of the scion. In some embodiments, the RNA cargo comprising the guide RNA linked to the engineered phloem mobility element is delivered to the rootstock and transported into the scion. In some embodiments, the guide RNA is produced in vitro. In some embodiments, the guide RNA is methylated in vitro, such as by an RNA methylase, to promote mobility. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. Delivery of the guide RNA can occur through the following non-exhaustive list: through use of an RNA spray comprising the guide RNA and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by application of a composition comprising the guide RNA onto a leaf after rubbing the leaf with 200 grit sandpaper with a dowel; by spraying onto a leaf very fine glass beads coated with a composition comprising the guide RNA; by injection of a composition comprising the guide RNA into the stem; by infiltration of the leaf with a composition comprising the guide RNA; by direct uptake in the roots of a composition comprising the guide RNA; or by biolistic delivery to leaves or other tissue with circular DNA expressing the guide RNA. In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and / or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA. In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and / or meristem. In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem.40MF-365821254Attorney Docket No.: 16536-20022.40In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump. In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the nuclease inhibitor comprises an RNase inhibitor. In some embodiments, delivery of the guide comprises biolistic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, shoot, stem, and / or meristem. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA. In some embodiments, the guide RNA is linked to an engineered phloem mobility element, wherein the engineered phloem mobility element increases transport of the guide RNA within the plant.

[0135] In some embodiments, the provided method of providing RNA cargo comprising genome editing reagents linked to an engineered phloem mobility element to the rootstock and / or scion of a plant, and thereby editing a genomic target in a scion. In some embodiments, comprises grafting the scion onto a rootstock expressing a Cas nuclease, wherein the rootstock comprises nucleic acid encoding the Cas nuclease linked to an engineered phloem mobility element; and delivering a guide RNA for the Cas nuclease to the scion by virus-mediated delivery. In some embodiments, the method further comprises transforming the rootstock with nucleic acid encoding the Cas nuclease prior to grafting. In some embodiments, the scion comprises a leaf, a shoot, a stem, and / or a meristem. In other aspects, provided herein are methods of editing a genomic target in the meristem of a plant comprising transforming the root of the plant with nucleic acid encoding a Cas nuclease; and delivering a guide RNA for the Cas nuclease to a leaf, a shoot, a stem, and / or meristem of the plant by virus-mediated delivery, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element. In some embodiments, the guide RNA is linked to an engineered phloem mobility element.

[0136] In some embodiments, a first genome editing reagent is provided to the plant as RNA cargo linked to an engineered phloem mobility element, and a second genome editing reagent is provided to the plant via a recombinant plant virus. In some embodiments, the second genome editing reagent is also linked to an engineered phloem mobility element. In some embodiments, the composition comprising the recombinant plant virus is infectious sap. In some embodiments, the second genome editing reagent is a gRNA. In some embodiments, the infectious sap is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious sap from the host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64). In some embodiments, the method 41MF-365821254Attorney Docket No.: 16536-20022.40further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious sap from the selected plants (see Mandal et al. Plant Dis. 2002, 9: 939-944; Mandal et al. J. Virol Meth. 2008, 149: 195-198; Laidlaw EPPO Bulletin 1987, 17:81-89; Sundaresha et al. Physiol Mol Biol Plants 2012, 18(4): 365-369; Mahas et al. Methods Mol Biol. 2019, 1917: 311-326; Mahmood et al. Viruses 2023, 15(2): 531). In some embodiments, the composition comprising the recombinant plant virus is infectious lysate. In some embodiments, the infectious lysate is provided by inoculating the leaves of a host plant with an infectious cDNA plasmid and collecting infectious lysate from the host plant (see Li & Hataya Virology J. 2019, 16:18; Tran et al. J. Virol Methods 2014, 201: 57-64). In some embodiments, the method further comprises selecting one or more intermediate host plants that are infected with viruses carrying intact cargo comprising the guide RNA, raising the selected plants, and collecting the infectious lysate from the selected plants. In some embodiments the virus is Foxtail Mosaic Virus (FoMV). In some embodiments, the plant is corn. In some embodiments, intermediate host plants that are highly infected with viruses are identified by detecting expression levels of viral coat protein, and / or detecting the presence of intact viral cargo. Several methods of detecting protein expression are known in the art, including but not limited to Western blots and ELISA assays (enzyme-linked immunosorbent assays). Several methods of detecting viral cargo are known in the art, including but not limited to PCR-based methods. In some embodiments, intermediate host plants that are highly infected with viruses are identified by measuring levels of viral coat protein-encoding mRNA in the intermediate host plants by RT-qPCR. In some embodiments, assaying the presence of intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant to confirm that no spontaneous mutations have accumulated in the cargo to be delivered. In some embodiments, assaying intact viral cargo comprises sequencing infectious cDNAs in the intermediate host plant, comparing the obtained sequence to the sequence of the provided infectious cDNA plasmid, and determining that the intact viral cargo is substantially identical or completely identical to the corresponding sequence of the provided infectious cDNA plasmid. In some embodiments, the infectious sap is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious sap from the tobacco leaves. In some embodiments, the infectious lysate is provided by performing leaf infiltration of tobacco leaves with bacteria comprising an infectious cDNA plasmid, and collecting infectious lysate from the tobacco leaves. Leaf infiltration with Agrobacterium is also referred to herein as “agroinfiltration”. Agroinfiltration includes but is not limited to 42MF-365821254Attorney Docket No.: 16536-20022.40syringe-based agroinfiltration and vacuum-based agroinfiltration. In syringe-based agroinfiltration, a composition comprising Agrobacterium is placed in a needleless syringe, which is placed against the underside of a leaf. The composition is then injected into the airspace within the leaf. In vacuum-based agroinfiltration, leaf tissue, leaves, or whole plants are submerged in a composition comprising Agrobacterium, which is contained within a vacuum chamber. Vacuum is applied, forcing air out of intercellular spaces within the leaves. Releasing the vacuum results in introduction of the composition into the leaves. In some embodiments, delivery of the guide RNA linked to the engineered phloem mobility element comprises direct leaf rub inoculation with infectious sap. In some embodiments, delivery of the guide RNA linked to the engineered phloem mobility element comprises direct leaf rub inoculation with infectious lysate.

[0137] In some embodiments, RNA cargo linked to the engineered phloem mobility element is transported by plant vascular system. In some embodiments, RNA cargo linked to the engineered phloem mobility element is transported to the scion through the xylem or the phloem. In some embodiments, RNA cargo linked to the engineered phloem mobility element is transported to the meristem.

[0138] In some embodiments, RNA cargo comprising or encoding the Cas nuclease and / or the guide RNA linked to an engineered phloem mobility element is transported by plant vascular system. In some embodiments, RNA cargo comprising or encoding the Cas nuclease and / or the guide RNA is transported to the scion through the xylem or the phloem. In some embodiments, RNA cargo comprising or encoding the Cas nuclease and / or the guide RNA is transported to the meristem. In some embodiments, RNA cargo comprising or encoding the Cas nuclease is translated in the meristem. In some embodiments, one or more meristematic cells is edited.

[0139] In some embodiments, the RNA cargo comprises a guide RNA linked to an engineered phloem mobility element, wherein the guide RNA linked to the engineered phloem mobility element is transported to the meristem of the plant scion, or is provided to the meristem of the plant scion directly. The guide RNA is imported into the meristem nuclei. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease into the meristem nuclei, the genome of the meristem nuclei is edited. Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes.

[0140] In some embodiments, methods provided herein allow for fast and modular editing of a multitude of plants, including elite lines, without the introduction of a transgene to the 43MF-365821254Attorney Docket No.: 16536-20022.40genome of the edited plant scion. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on rootstock plants providing the Cas nuclease, and different guide RNAs, each linked to an engineered phloem mobility element described herein, can be delivered to the different plant scions. Because there isn’t a different transgene being inserted into a different location in each plant scion, this allows for direct comparison of the results of providing different guide RNAs, including but not limited to comparison of efficiency of method of delivery, editing efficiency of different guide RNAs, and phenotypic changes as a result of edits induced by different guide RNAs. The provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.

[0141] Additionally, in some embodiments, the provided methods allow for a reduced number of required transformation events. The rootstock providing the Cas nuclease can be used with a wide variety of delivered guide RNAs (for example, with each linked to an engineered phloem mobility element), increasing the modularity of the editing system.E. Uptake of RNA cargo by roots

[0142] In some embodiments, provided herein are methods of providing an RNA cargo linked to an engineered phloem mobility element to a plant via a root of the plant. In some embodiments, the RNA cargo linked to the phloem mobility element is provided to a rootstock. In some embodiments, a scion is grafted onto the rootstock. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is provided to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. In some embodiments, the RNA cargo is transported via the vascular system of the plant. In some embodiments, the RNA cargo is transported to the meristem of the plant. In some embodiments, the RNA cargo is transported to the plant scion. In some embodiments, the RNA cargo is transported from the rootstock to the scion through the graft junction. In some embodiments, the RNA cargo is transported through the xylem or the phloem. In some embodiments, transport of the RNA cargo is increased when linked to an engineered phloem mobility element described herein.

[0143] In some embodiments, the present application provides methods of editing a genomic target in a plant meristem comprising providing a plant comprising nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding a Cas nuclease is linked to an engineered phloem44MF-365821254Attorney Docket No.: 16536-20022.40mobility element, and delivering to the root of the plant a guide RNA for the Cas nuclease, optionally wherein the guide RNA is also linked to an engineered phloem mobility element. In some embodiments, the plant comprising the nucleic acid encoding a Cas nuclease is a rootstock. In some embodiments, a scion is grafted onto the rootstock. In some embodiments, the genomic editing reagents are provided to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. In some embodiments, the Cas nuclease is delivered to the plant root by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. The plant provides nucleic acid encoding a Cas nuclease to the plant vascular system. In some embodiments, the nucleic acid encoding a Cas nuclease is linked to an engineered phloem mobility element. The linkage of the engineered phloem mobility element to nucleic acid encoding the Cas nuclease results in the nucleic acid encoding the Cas nuclease being transported to cells of the meristem of the scion through the plant vascular system. In some embodiments, the nucleic acid encoding the Cas nuclease is transported from the rootstock to the scion through the graft junction. In some embodiments, RNA encoding the Cas nuclease linked to an engineered phloem mobility element and / or the guide RNA linked to an engineered phloem mobility element is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease linked to an engineered phloem mobility element and / or the guide RNA linked to an engineered phloem mobility element is transported through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported to the meristem, wherein the Cas nuclease and / or the guide RNA is translated in the meristem. Nucleic acid encoding the Cas nuclease is translated in the cytosol of cells of the scion meristem and imported into meristem nuclei.

[0144] In some embodiments, the guide RNA linked to the engineered phloem mobility element is delivered to the roots. In some embodiments, the guide RNA is delivered via direct uptake in the roots. In some embodiments, the guide RNA is delivered to the plant by infection with Agrobacterium rhizogenes (also known as Rhizobium rhizogenes), producing a plant with transgenic hairy roots. In some embodiments, the guide RNA is injected into the roots. In some embodiments, the guide RNA is produced in vitro. In some embodiments, the guide RNA is methylated in vitro, such as by an RNA methylase, to promote mobility. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. Delivery of the guide RNA can occur through the following non-exhaustive list: through use of an RNA spray comprising the guide RNA and a simple surfactant (see, e.g., U.S. Pat. No. 9,121,022); by injection of a composition comprising the guide RNA into the stem; by direct uptake in the roots of a 45MF-365821254Attorney Docket No.: 16536-20022.40composition comprising the guide RNA; or by biolistic transformation of roots or other tissue with circular DNA expressing the guide RNA. The guide RNA is transported to the meristem of the plant, and is imported into the meristem nuclei. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease into the meristem nuclei, the genome of the meristem nuclei is edited. In some embodiments, upon import of the guide RNA for the Cas nuclease into the meristem nuclei where the Cas nuclease is expressed, the genome of the meristem nuclei is edited Edits made in the scion meristem are heritable as the meristem nuclei will form the reproductive tissues of the plant, including the gametes. The guide RNA is transported to the meristem of the plant, and is imported into the meristem cell. Upon import of both the Cas nuclease and the guide RNA for the Cas nuclease into the meristem cell, the genome of the meristem cell is edited. Edits made in the scion meristem cell are heritable as the meristem cell will form the reproductive tissues of the plant, including the gametes.

[0145] In some embodiments, provided methods for editing a grafted scion allow for fast and modular editing of a multitude of plants, including elite lines, without the introduction of a transgene to the edited genome. Edits can be made in any plant that can be grafted onto a provided rootstock, including plant species that are intractable to transformation. Many scions from the same line can be grafted on the rootstock, allowing for direct comparison of the results of providing different guide RNAs each linked to an engineered phloem mobility element described herein, including but not limited to comparison of efficiency of method of delivery, editing efficiency of different guide RNAs, and phenotypic changes as a result of edits induced by different guide RNAs. The provided methods will enlarge the capacity of a plant editing pipeline to make edits and observe the resulting phenotypes in genetic backgrounds of commercial relevance.

[0146] In some embodiments, the provided methods for editing a plant transformed with Agrobacterium rhizogenes allow for a fast and modular introduction of heritable edits. A strain of Agrobacterium is developed that comprises the Cas nuclease linked to an engineered phloem mobility element, and this strain can be used to infect and transform a variety of plants. This results in a variety of plants to which a guide RNA, also linked to an engineered phloem mobility element, can be delivered to produce heritable edits in the plant meristem. This method does not require any additional generations between the transformation with Agrobacterium and the production of an edited genomic target, and is thus an improvement on current editing techniques. This method does not require any additional generations between the transformation with Agrobacterium and the production of heritable edits, and is thus an improvement on current editing techniques.46MF-365821254Attorney Docket No.: 16536-20022.40F. Grafting

[0147] In some embodiments, the method provided herein comprise delivering RNA cargo comprising one or more genome editing reagents linked to an engineered phloem mobility element to a rootstock and / or a scion, and editing a grafted scion. The present disclosure utilizes grafting systems and their vascular mobility in some embodiments to accomplish delivery of genome editing reagents (e.g., a Cas nuclease and / or a gRNA for the Cas nuclease) via the plant vascular system to the meristem of a plant, wherein editing of a meristem cell of the plant can occur. . Grafting can be performed, for example, by inserting one or more cut scion stems into a cut of a rootstock stem, wherein the vascular tissue of the scion stem and the rootstock stem are substantially aligned. A stabilization device may be used.

[0148] A successful graft exhibits a continuous vascular system from rootstock to scion, including transmission through a graft junction. RNAs and / or endonucleases expressed in the rootstock and linked to engineered phloem mobility elements described herein, in some embodiments encoding genome editing reagents, enter the phloem and transit to the shoot apical meristem of the scion. The RNAs and / or endonucleases are imported into cells of the meristem and are processed into functional RNPs, which are able to modify the genome of the meristem of the plant scion. The present disclosure provides methods of providing RNA cargo comprising genome editing reagents and linked to an engineered phloem mobility element to a rootstock and / or a scion of a plant, and thereby editing the genome of a transgene-free plant scion, wherein the plant scion genome does not contain DNA encoding reagents for genomic modification.

[0149] A plant scion transformed through the present methods of genomic editing does not contain transgenes encoding the reagents for genomic modification. The plant scion must be able to be grafted onto a transformed rootstock, but it is not necessary that the plant scion itself be transformable. This widens the possibility of species that can be edited through the present disclosure. Additionally, many plants can be grafted onto the same variety of rootstock, thus speeding development of genomically edited scions.G. Configurations of Agrobacterium vector systems

[0150] The vector systems (e.g., Agrobacterium rhizogenes vector systems) of the present disclosure may be modified as desired, including with processing elements, in order to optimize efficiency of the transfection and promote transport of an RNA cargo linked to an engineered phloem mobility element within the vascular system of a plant.47MF-365821254Attorney Docket No.: 16536-20022.40

[0151] In some embodiments, a vector provided herein comprises an RNA cargo, an engineered phloem mobility element, and a linker. In some embodiments, the RNA cargo comprises multiple cargo elements, for example, multiple cistrons. In some embodiments, the RNA cargo comprises one or more genome editing reagents. In some embodiments, RNA cargo linked to an engineered phloem mobility element is provided to the plant in a single vector. In some embodiments, RNA cargo linked to an engineered phloem mobility element is provided to the plant in multiple vectors. In some embodiments, the RNA cargo comprises an RNA encoding a Cas nuclease and a guide RNA, wherein the RNA cargo is linked to an engineered phloem mobility element and provided to the plant in a single vector. In some embodiments, a first RNA cargo comprises an RNA encoding a Cas nuclease, wherein the first RNA cargo is linked to a first engineered phloem mobility element and provided to the plant via a first vector, and a second RNA cargo comprises a guide RNA for the Cas nuclease, wherein the second RNA cargo is linked to a second engineered phloem mobility element and provided to the plant via a second vector.H. CRISPR-Cas systems

[0152] In some embodiments, the RNA cargo comprises one or more components of a CRISPR / Cas system. In some embodiments, the RNA cargo is linked to an engineered phloem mobility element and delivered to the plant, wherein the one or more components of the CRISPR / Cas system are transported to a meristem cell of the plant. In some embodiments, the meristem cell is edited.

[0153] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas systems, or CRISPR systems, are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Casl2a (“Cpfl”)) to cleave foreign DNA. In a typical CRISPR / Cas system, a Cas endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. In microbial hosts, CRISPR loci encode both Cas endonucleases and “CRISPR arrays” of the non-coding RNA elements that determine the specificity of the CRISPR-mediated nucleic acid cleavage.

[0154] The genomic DNA sequence targeted for editing or modification must generally be adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas48MF-365821254Attorney Docket No.: 16536-20022.40endonuclease; however, PAM sequences are short and relatively non-specific, appearing throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), 5'-NNGRRT or 5'-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5'-NNNGATT (Neisseria meningitidis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleaving of the target DNA at a location three nucleotides upstream from (5' from) the PAM site. Casl2a (Cpfl) CRISPR systems cleave the target DNA adjacent to a short T-rich PAM sequence, e.g., 5'-TTN, in contrast to the G-rich PAM sequences identified for Cas9 systems. Examples of Casl2a PAM sequences include those for the naturally occurring Acidaminococcus sp. BV3L6 Cpfl (AsCpfl) and Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl) TTTV, where V can be A, C, or G. In some instances, Casl2a can also recognize a 5'-CTA PAM motif. Other examples of potential Casl2a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Various methods (including in silico and / or wet lab methods) for identification of the appropriate PAM sequence are known in the art and are routine, and any convenient method can be used. A PAM sequence can be identified using a PAM depletion assay. Casl2a cleaves the target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. Cell 2015, 163: 759-771.I. Nucleases

[0155] Two classes (1 and 2) of CRISPR systems have been identified across a wide range of bacterial hosts. The well characterized class 2 CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class 2 CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”), see Guide RNA below. The Casl2a (“Cpfl”) CRISPR system includes the type49MF-365821254Attorney Docket No.: 16536-20022.40V endonuclease Casl2a (also known as “Cpfl”). Casl2a nucleases are characterized as having only a RuvC nuclease domain, in contrast to Cas9 nucleases which have both RuvC and HNH nuclease domains. Casl2a nucleases are generally smaller proteins than Cas9 nucleases and can function with a smaller guide RNA (e.g., a crRNA having at least one spacer flanked by direct repeats), which are practical advantages in that the nuclease and guide RNAs are more economical to produce and potentially more easily delivered to a cell. Examples of Casl2a nucleases include AsCasl2a or “AsCpfl” (from Acidaminococcus sp.) and LbCasl2a or “LbCpfl” (from Lachnospiraceae bacteria). In contrast to Cas9 type CRISPR systems, Casl2a-associated (“Cpfl”-associated) CRISPR arrays have been reported to be processed into mature crRNAs without the requirement of a tracrRNA, i.e., the naturally occurring Casl2a (Cpfl) CRISPR system was reported to require only the Casl2a (Cpfl) nuclease and a Casl2a crRNA to cleave the target DNA sequence; see Zetsche et al. Cell 2015, 163: 759-771; U.S. Pat. No. 9,790,490. Casl2f is another type of nuclease in the Casl2 family (alongside Casl2a, Casl2b, etc.), and its compact size makes it an excellent candidate for being included in a viral vector system.

[0156] It is understood that for all systems, the use of a nuclease activity for cutting DNA followed by repair by the endogenous cell machinery is one solution to generate useful mutants. The nuclease activity can be eliminated or altered, as in dCas (“dead” Cas, i.e., Cas with no nuclease functionality) or nCas (“nickase” Cas, i.e., Cas that makes single- stranded breaks rather than double- stranded breaks), TALE (TAL-effector), or ZF (zinc finger) versions of the polypeptides. Inactivated nucleases can be useful for targeting the desired DNA sequence, while editing can be performed by nucleobase editors attached to the altered nucleases. Examples are included in W02018176009 and US Patent No. 10,113,163, incorporated herein by reference.

[0157] Useful CRISPR-based RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see W02018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337). Useful RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see W02018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337). “Casl2” is used herein to refer to any Casl2 protein, including but not limited to Casl2f, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see W02018176009), Casl2h, Casl2i (see Yan et al.50MF-365821254Attorney Docket No.: 16536-20022.40Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337. In some embodiments, the Cas nuclease is a nuclease selected from the group consisting of Cas9, Casl2f, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j. In some embodiments, the Cas nuclease is a nuclease selected from the group consisting of Cas9, Casl2f, Casl2a (Cpfl), any Mini Cas, Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j, TnpB, IscB, and any omegaRNA. In some embodiments, the Cas nuclease is a Cas nickase. In some embodiments, the Cas nuclease is a Cas9 nuclease or a Cas 12 nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease or a Cas 12 nuclease. In some embodiments, the Cas nickase is a Cas9 nickase or a Casl2 nickase. In some embodiments, the Cas nickase comprises mutation in one or more nuclease active sites. In some embodiments, the Cas nuclease is associated with a reverse transcriptase.

[0158] It is understood that for all systems, the use of a nuclease activity for cutting DNA followed by repair by the endogenous cell machinery is one solution to generate useful mutants. The nuclease activity can be eliminated or altered, as in dCas (“dead” Cas, i.e., Cas with no nuclease functionality) or nCas (“nickase” Cas, i.e., Cas that makes single- stranded breaks rather than double- stranded breaks), TALE (TAL-effector), or ZF (zinc finger) versions of the polypeptides. Inactivated nucleases can be useful for targeting the desired DNA sequence, while editing can be performed by nucleobase editors attached to the altered nucleases. Examples are included in W02018176009 and US Patent No. 10,113,163, incorporated herein by reference.

[0159] Useful CRISPR-based RNA-guided nuclease systems have been described and are known from the literature, including but not limited to Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see W02018176009), Casl2h, Casl2i (see Yan et al. Science 2019, 363(6422): 88-91) and Casl2j (Pausch et al. Science 2020, 369(6501): 333-337). “Cas 12” is used herein to refer to any Cas 12 protein, including but not limited to Cas 12a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3 (see WO2018176009), Casl2h, Casl2i (see Yanetal. Science 2019, 363(6422): 88-91) and Casl2j (Pausch etal. Science 2020, 369(6501): 333-337. In some embodiments, the Cas nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j. In some embodiments, the Cas nuclease is selected from the group consisting of Cas9, Casl2a (Cpfl), a Mini Cas, Casl2e (CasX), Casl2d (CasY), C2cl, C2c2, C2c3, Casl2h, Casl2i, and Casl2j. In some embodiments, the Cas nuclease is a Cas nickase. In some embodiments, the Cas nuclease is a Cas9 nuclease or a Casl2 nuclease. In some 51MF-365821254Attorney Docket No.: 16536-20022.40embodiments, the Cas nickase is a Cas9 nickase or a Casl2 nickase. In some embodiments, the Cas nickase comprises mutation in one or more nuclease active sites. In some embodiments, the Cas nuclease is associated with a reverse transcriptase.

[0160] In some embodiments, the RNA-guided nuclease of the present disclosure is an evolutionary progenitor of Cas endonuclease(s). In some embodiments, the RNA-guided nuclease is a member of the OMEGA system (Obligate Mobile Element Guided Activity system). In some embodiments, the RNA-guided nuclease is a TnpB nuclease and / or an IscB nuclease.

[0161] In embodiments of the present disclosure, the term “Cas enzyme” includes all RNA-guided nucleases.

[0162] In some embodiments, the gRNA and the Cas nuclease form a complex and introduce a single- or double- stranded break in the sequence of the genomic target. In some embodiments, the viral vector comprising the gRNA further comprises a Casl2f nuclease. In some embodiments, the viral vector comprising the gRNA does not comprise the Cas nuclease. In some embodiments, the viral vector comprising the Cas nuclease does not comprise the gRNA. In some embodiments, the viral vector comprising the gRNA further comprises a Mini Cas.

[0163] In a phenomenon termed “codon bias”, different organisms use specific codons more often than synonymous codons to encode for the same amino acid. Furthermore, efficiency of mRNA translation can be correlated with the use of the preferred codons over less frequently used codons. A nucleic acid can therefore be optimized for expression in a desired host by replacing codons less frequently used in that host with those more frequently used in the host. Codon bias varies across species, as well as across wider phylogenetic distance. Codon usage tables are known in the art (see, e.g., the “Codon Usage Database” at www[dot]kazusa[dot]or[dot]jp[forward slash]codon) and these tables can be adapted in a number of ways, as shown in Nakamura et al. (Nucl Acids Res 2000, 28: 292). Computer algorithms may also be used for codon optimization of a particular sequence for expression in a desired host, such as Gene Forge (Aptagen; Jacobus, PA). For use in plants, see e.g., Campbell and Gowri (Plant Physiol 1990, 92: 1-11) and Murray et al. (Nucl Acids Res 1989, 17: 477-498).

[0164] A Cas nuclease is encoded by a nucleic acid. In one embodiment, the nucleic acid encoding the Cas nuclease is codon-optimized for use in a species of plant. In some embodiments, the Cas nuclease is codon-optimized for expression in dicots. In some embodiments, the Cas nuclease is codon-optimized for expression in soybean. In some embodiments, the Cas nuclease is codon-optimized for expression in monocots. In some 52MF-365821254Attorney Docket No.: 16536-20022.40embodiments, the Cas nuclease is codon-optimized for expression in corn. In some embodiments, the Cas nuclease is codon-optimized for expression in wheat. In some embodiments, the Cas nuclease is fused to a nuclear localization signal (NLS). CRISPR nuclease fusion proteins containing nuclear localization signals and codon-optimized for expression in maize are disclosed in U.S. patent application Ser. No. 15 / 120,110, published as U.S. Patent Application Publication 2017 / 0166912, national phase application claiming priority to PCT / US2015 / 018104 (published as WO / 2015 / 131101 and claiming priority to U.S. Provisional Patent Application 61 / 945,700), incorporated herein by reference.

[0165] The nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element. In some embodiments, the nucleic acid encoding at least one guide RNA and the nucleic acid encoding the Cas nuclease are linked to one or more engineered phloem mobility elements. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion by the plant vascular system. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are transported from the scion to the rootstock through the phloem. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are transported from the rootstock to the scion through the plasmodesmata. In some embodiments, RNA encoding the Cas nuclease and at least one guide RNA are translated in the cytosol of a meristem cell. In some embodiments, translation of the RNA encoding the Cas nuclease and at least one guide RNA in the cytosol of a meristem cell results in editing of the genome of the meristem cell. In some embodiments, the meristem cell is on the plant scion.

[0166] In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a promoter. For use in plants, useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter). In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a constitutive promoter. Examples of constitutive promoters include a CaMV 35S promoter as disclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Pat. No. 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Pat. No. 7,151,204, an opaline synthase (NOS) and octopine synthase (OCS) promoter from Agrobacterium tumefaciens, and a ubiquitin promoter. In some embodiments, the nucleic acid encoding the Cas enzyme is linked to an inducible promoter. An “inducible” promoter is a promoter that initiates transcription in response to an 53MF-365821254Attorney Docket No.: 16536-20022.40environmental stimulus such as heat, cold, drought, light, or other stimuli, such as wounding or chemical application. Examples of inducible promoters include, but are not limited to, those described in U.S. Pat. No. 6,294,714 (light inducible promoters), U.S. Pat. No. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), and U.S. Pat. No. 6,175,060 (phosphorus deficiency inducible promoters). In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a promoter selected from the group consisting of promoters active in roots and promoter active in phloem companion cells.

[0167] In some embodiments, the promoter is a constitutive promoter, optionally wherein the constitutive promoter is a ubiquitin promoter. In some embodiments, the promoter is selected from the group consisting of a promoter from a Arabidopsis WRKY6 gene, a promoter from a chickpea WRKY31 gene, a promoter from a carrot MYB113 gene, a promoter from a com GLU1 gene, a promoter from a strawberry RB7-type TIP-2 gene, a promoter from a banana TIP2-2 gene, a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabaceaen FORI gene, a rice tungro bacilliform vims promoter, an RmlC-like cupins superfamily protein promoter, a Commelina yellow mottle vims promoter, a wheat dwarf virus promoter, a sucrose synthase promoter, a glutamine synthetase promoter, a phloem-specific isoform of plasmamembrane H-F-ATPase promoter, a JMJ18 promoter, and a phloem protein 2 (PP2) promoter, or the promoter of an orthologous gene thereof. In some embodiments, the promoter is active in roots and / or phloem companion cells. In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a promoter selected from the group consisting of promoters active in roots and promoter active in phloem companion cells.

[0168] In some embodiments, the promoter active in roots is the promoter of a gene selected from the group consisting of Arabidopsis thaliana WRKY6 or orthologous genes thereof, chickpea WRKY31 or orthologous genes thereof, carrot MYB113 or orthologous genes thereof, corn GLU1 or orthologous genes thereof, strawberry RB7-type TIP-2 or orthologous genes thereof, and banana TIP2-2 or orthologous genes thereof. Additional suitable root promoters are provided in the RGPDB database (database of root-associated genes and promoters in maize, soybean, and sorghum) as described in Moisseyev et al. Database, 1-7 (2020). In some embodiments, the promoter active in phloem companion cells is selected from the group consisting of a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabaceaen FORI gene (Noll et al. Plant Mol Biol 2007, 65(3): 285-294), a rice tungro bacilliform virus promoter (Yin et al. Plant J 1997, 12(5): 1179-1188), an RmlC-like cupins superfamily protein promoter (CN102002498B), a Commelina yellow mottle virus promoter (Medberry et al., Plant Cell 1992, 4: 185-192), a wheat dwarf virus promoter 54MF-365821254Attorney Docket No.: 16536-20022.40(W02003060135A2), a sucrose synthase promoter (Yang and Russell PNAS 1990, 87: 4144-4148), a glutamine synthetase promoter (Edwards et al. PNAS 1990, 87: 3459-3463), a phloemspecific isoform of plasmamembrane H+-ATPase promoter (DeWitt et al. Plant J. 1991, 1(1): 121-128), a JmjC domain-containing protein 18 (JMJ18) promoter (Yang et al., PLoS Genet 2012, 8(4): el002664), and a phloem protein 2 (PP2) promoter (US5495007A).

[0169] The nucleic acid encoding the Cas nuclease and / or at least one guide RNA may be transcribed in a rootstock. In some embodiments, the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element. In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the engineered phloem mobility element is located 5’ of the nucleic acid encoding the Cas nuclease. In some embodiments, engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the engineered phloem mobility element is located both 5’ and 3’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the nucleic acid encoding the Cas nuclease and / or guide RNA is intended to be transcribed in a cell of the rootstock, transported through the graft junction to the scion, and translated inside a scion meristem cell. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported from the rootstock to the scion by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease is translated in the scion. In some embodiments, RNA encoding the Cas nuclease is transported to the meristem, wherein the Cas nuclease and / or the guide RNA is translated in the meristem. As such, the nucleic acid encoding the Cas nuclease is typically embedded within an mRNA component. A 5’ cap and polyA tail are also useful in stabilizing the RNA. A 5’ UTR has translation initiation sequences upstream of the Cas coding sequence. A 5’ UTR can also have small upstream open reading frames that affect translation (Jorgensen and Dorantes-Acosta, Front. Plant Sci 2012, 3:191). For example, an mRNA can comprise a 5’ UTR comprising a 7 -methylguanosine cap at its 5’ terminus followed by an untranslated sequence and terminated by the translation initiation codon of the coding sequence (e.g., the Cas coding sequence).

[0170] The nucleic acid encoding the Cas nuclease and / or at least one guide RNA is intended to be transcribed in the rootstock. In some embodiments, the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element. In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas 55MF-365821254Attorney Docket No.: 16536-20022.40nuclease. In some embodiments, the engineered phloem mobility element is located 5’ of the nucleic acid encoding the Cas nuclease. In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas nuclease. The nucleic acid encoding the Cas nuclease and / or guide RNA is intended to be transcribed in a cell of the rootstock, transported through the graft junction to the scion, and translated inside a scion meristem cell. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported from the rootstock to the scion by plant vascular system. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported to the scion through the xylem or the phloem. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is translated in the scion. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported to the meristem, wherein the Cas nuclease and / or the guide RNA is translated in the meristem. In some embodiments, RNA encoding the Cas nuclease and / or the guide RNA is transported to the meristem cell, wherein the Cas nuclease and / or the guide RNA is translated in the meristem cell. As such, the nucleic acid encoding the Cas nuclease and / or the guide RNA is typically embedded within an mRNA component. A 5’ cap and polyA tail are also useful in stabilizing the RNA. A 5’ UTR has translation initiation sequences upstream of the Cas coding sequence. A 5’ UTR can also have small upstream open reading frames that affect translation (Jorgensen and Dorantes-Acosta, Front. Plant Sci 2012, 3:191). For example, an mRNA can comprise a 5’ UTR comprising a 7 -methylguanosine cap at its 5’ terminus followed by an untranslated sequence and terminated by the translation initiation codon of the coding sequence (e.g., the Cas coding sequence).

[0171] The nucleic acid encoding the Cas nuclease can be optimized to increase nuclease activity and editing efficiency. In some embodiments, the nucleic acid encoding the Cas enzyme is linked to a nuclear localization signal (NLS), such as the NLS from SV40. Various NLSs, including those that bind to the major groove and / or the minor groove of an importin protein, are well known in the art, as in Kosugi et al. (J Biol Chem 2009, 284(1): 478-485). In some embodiments, the nucleic acid encoding the Cas nuclease is fused to a cell penetrating peptide (CPP), such as octa-arginine or nona- arginine or a homoarginine 12-mer oligopeptide, or a CPP disclosed in the database of cell-penetrating peptides CPPsite 2.0, publicly available at webs[dot]iiitd[dot]edu[dot]in / raghava / cppsite / (Kardani and Bolhassani J Mol Biol 2021, 433(11): 166703). In some embodiments, the nucleic acid encoding the Cas enzyme further 56MF-365821254Attorney Docket No.: 16536-20022.40comprises a terminator. By “terminator” is meant a DNA segment near the 3' end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA. Such a 3' element is also sometimes referred to as a “3 '-untranslated region” or “3'-UTR” or a “polyadenylation signal”. Non-limiting embodiments of terminators functional in eukaryotic cells include a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3' element from an Agrobacterium sp. Gene, a 3' element from a non-human animal gene, a 3' element from a human gene, and a 3' element from a plant gene, wherein the 3' element terminate transcription of an RNA transcript located immediately 5' to the 3' element. Useful 3' elements include: Agrobacterium tumefaciens nos 3', tml 3', tmr 3', tins 3', ocs 3', and tr7 3' elements disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference; 3' elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose- 1,6-biphosphatase genes from wheat (Tri li cum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza sativa), disclosed in U.S. Patent Application Publication 2002 / 0192813 Al, incorporated herein by reference; in some embodiments, the terminator is selected from the group consisting of CaMV 35S terminator, Atug7 terminator, NOS terminator, Act2 terminator, MAS terminator, tomato ATPase terminator, rbcSC3 terminator, potato H4 terminator, rbcSE9 terminator, GILT terminator, ALB terminator, API terminator, HSP terminator, and OCS terminator , as referenced in Hassan et al. (Trends Plant Sci 2021, 26: 1133-1152). In some embodiments, the nucleic acid encoding the Cas enzyme further comprises one or more introns. In some embodiments, the nucleic acid encoding the Cas enzyme further comprises one or more transcriptional enhancers. In some embodiments, the one or more transcriptional enhancers comprise one or more bacterial octopine synthase (OCS) enhancers (U.S. Patent No. 11,198,885). In one embodiment, the nucleic acid encoding the Cas enzyme further comprises a triple OCS enhancer (U.S. Patent No. 11,198,885). In some embodiments, the nucleic acid encoding the Cas enzyme further comprises a 5’ UTR comprising a translational enhancer. In some embodiments, the nucleic acid encoding the Cas enzyme further comprises a Kozak sequence endogenous to the scion species at the translation start codon. In some embodiments, the nucleic acid encoding the Cas enzyme further comprises nuclear localization signals flanking the coding sequence of the Cas enzyme.

[0172] In some embodiments, a viral vector or viral vector system, for example, an Agrobacterium vector, is delivered to a plant that already overexpresses a Cas nuclease. In some embodiments, the plant overexpresses Cas9. This overexpression of a Cas nuclease may 57MF-365821254Attorney Docket No.: 16536-20022.40be the result of gene editing conducted before the viral infection of the plant, and this overexpression can be produced through a variety of gene editing methods known in the art, not limited to viral infection of a viral vector carrying the Cas nuclease.J. Guide RNAs

[0173] CRISPR-based RNA-guided nuclease systems typically require an effector polypeptide and one or more guide RNAs (gRNAs). The guide RNAs are generally made up of an effector-binding region and a target DNA recognition region, and in some embodiments include tracrRNAs. A “trans-activating crRNA” or “tracrRNA” is a trans-encoded small RNA that is partially homologous to repeats within a CRISPR array. At least in the case of Cas9 type CRISPR systems, both a tracrRNA and a crRNA are required for the CRISPR array to be processed and for the nuclease to cleave the target DNA sequence. In contrast, Casl2a type CRISPR systems have been reported to function without a tracrRNA, with the Cas 12a CRISPR arrays processed into mature crRNAs without the requirement of a tracrRNA; see Zetsche et al. Cell 2015, 163: 759-771 and U.S. Pat. No. 9,790,490. The Cas9 crRNA contains a “spacer sequence”, typically an RNA sequence of about 20 nucleotides (in various embodiments this is 20, 21, 22, 23, 24, 25, or up to about 30 contiguous nucleotides in length) that corresponds to (e.g., is identical or nearly identical to, or alternatively is complementary or nearly complementary to) a target DNA sequence of about equivalent length. The Cas9 crRNA also contains a region that binds to the Cas9 tracrRNA to form a partially double-stranded structure which is cleaved by RNase III, resulting in a crRNA: tracrRNA hybrid or duplex. The crRNA: tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence; in some examples, a tracrRNA and crRNA (e.g., a crRNA including a spacer sequence) can be included in a chimeric nucleic acid referred to as a “single guide RNA” (sgRNA).

[0174] As used herein “guide RNA” or “gRNA” refers to a nucleic acid that comprises or includes a nucleotide sequence (sometimes referred to a “spacer sequence”) that corresponds to (e.g., is identical or nearly identical to, or alternatively is complementary or nearly complementary to) a target DNA sequence (e.g., a contiguous nucleotide sequence that is to be modified) in a genome; the guide RNA functions in part to direct the CRISPR nuclease to a specific location on the genome. In embodiments, a gRNA is a CRISPR RNA (“crRNA”), such as the engineered Cas 12a crRNAs described in this disclosure. For nucleases (such as a Cas9 nuclease) that require a combination of a trans-activating crRNA (“tracrRNA”) and a crRNA58MF-365821254Attorney Docket No.: 16536-20022.40for the nuclease to cleave the target nucleotide sequence, the gRNA can be a tracrRNA:crRNA hybrid or duplex, or can be provided as a single guide RNA (sgRNA). At least 16 or 17 nucleotides of gRNA sequence corresponding to a target DNA sequence are required by Cas9 for DNA cleavage to occur; for Casl2a (Cpfl) at least 16 nucleotides of gRNA sequence corresponding to a target DNA sequence are needed to achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence corresponding to a target DNA sequence were reported necessary for efficient DNA cleavage in vitro; see Zetsche et al. Cell 2015, 163: 759-771. Casl2a (Cpfl) endonuclease and corresponding guide RNAs and PAM sites are disclosed in U.S. Pat. No. 9,790,490, which is incorporated herein by reference in its entirety and particularly for its disclosure of DNA encoding Casl2a (Cpfl) endonucleases and guide RNAs and PAM sites. In practice, guide RNA sequences are generally designed to contain a spacer sequence of between 17-24 contiguous nucleotides (frequently 19, 20, or 21 nucleotides) with exact complementarity (e.g., perfect base-pairing) to the targeted gene or nucleic acid sequence; guide RNAs having spacers with less than 100% complementarity to the target sequence can be used (e.g., a gRNA with a spacer having a length of 20 nucleotides and between 1-4 mismatches to the target sequence), but this can increase the potential for off-target effects. The design of effective guide RNAs for use in plant genome editing is disclosed in U.S. Patent Application Publication 2015 / 0082478 Al, the entire specification of which is incorporated herein by reference. Chemically modified sgRNAs have been demonstrated to be effective in Cas9 genome editing; see, for example, Hendel et al. Nature Biotechnol., 2015, 33:985-991.

[0175] Guide RNA(s) can be part of the same RNA (mRNA) capable of expressing the Cas nuclease. In one embodiment, one or more guide RNAs are flanked by direct repeats (DR) of the CRISPR array from which the Cas effector polypeptide was first isolated. In some embodiments, the two or more guide RNAs are each flanked by a direct repeat. For example, a translated and expressed active Cas 12a nuclease can process the DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2a nuclease can process Casl2a DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2e nuclease can process Casl2e DR-flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2i nuclease can process Casl2i DR- flanked spacers of the mRNA to make guide RNAs. In certain embodiments, a translated and expressed active Casl2j nuclease can process Casl2j DR-flanked spacers of the mRNA to make guide RNAs. In alternative embodiments, a guide RNA suitable for matching an expressed effector polypeptide is flanked 59MF-365821254Attorney Docket No.: 16536-20022.40by processing elements, so that functional guide RNAs are excised inside the cells. Exemplary processing elements include hammerhead ribozymes, Csy4, and tRNAs (see Mikami et al. Plant Cell Physiol. 2017, 58(11): 1857-1867; and US Patent No. 10,308,947). Ribozymes can autocatalytically cleave the RNA to release the guide RNA from a polycistronic transcript and / or remove additional 5’ or 3’ sequence around the guide RNA. tRNAs are processed by elements of the cell’s endogenous tRNA system, such as RNase P, RNase Z, and RNase E, and tRNA sequences or pre-tRNA sequences can also be used to release a guide RNA flanked by processing elements from a polycistronic transcript and / or remove additional 5’ or 3’ sequence around the guide RNA. In some embodiments, the nucleic acid encoding the guide RNA and the engineered phloem mobility element is located between two ribozyme sequences. In some embodiments, each of the ribozyme sequences is independently selected from the group consisting of a hammerhead ribozyme sequence, a HDV ribozyme sequence, a Csy4 sequence, and a tRNA-derived sequence. In some embodiments, the nucleic acid encoding the guide RNA and the engineered phloem mobility element further comprises a hammerhead ribozyme sequence 5’ to the nucleic acid encoding the guide RNA and the engineered phloem mobility element, and a HDV ribozyme 3’ to the nucleic acid encoding the guide RNA and the engineered phloem mobility element. In some embodiments, a guide RNA is encoded by a nucleic acid. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease and / or 3’ of the nucleic acid encoding the guide RNA. In some embodiments, the engineered phloem mobility element is located 5’ of the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease and / or 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas9 nuclease or Casl2 nuclease and / or 3’ of the nucleic acid encoding the guide RNA. In some embodiments, the plant further comprises a nucleic acid encoding a detectable marker linked to the engineered phloem mobility element, optionally wherein the nucleic acid encoding the engineered phloem mobility element is located 3’ or 5’ of a nucleic acid encoding the Cas nuclease.

[0176] In some embodiments, the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target. In some embodiments, the nucleic acid encoding the guide RNA and the engineered phloem mobility element further comprises a terminator. In some embodiments, the terminator is a U6 terminator.

[0177] In some embodiments, the guide RNA comprises a 5-methylcytosine group.60MF-365821254Attorney Docket No.: 16536-20022.40

[0178] In some embodiments, the present invention comprises a guide RNA or guide RNA(s) which have chemical modifications. Chemical modifications are made to RNA molecules which then alter at least one of the four canonical ribonucleotides: A, U, C, and G. These modifications can be natural or unnatural and refer to a chemical moiety or portions of a chemical moiety which are not found in the unmodified canonical ribonucleotides. Alternative bases can include but are not limited to 2-thiouridine, 4-thiorudine, 2-aminoadenosine, 7-deazaguanosine, inosine, 5-methylcytidine, 5-aminoallyluridine, and 5-methyluridine. Either independently or additionally, a guide RNA which comprises any backbone or inter-nucleotide linkage other than a natural phosphodiester linkage is a chemically modified guide RNA. Alternative phosphodiester linkages can include but are not limited to an alkylphosphonate, a phosphonocaboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phoshporodithioate linkage. Either independently or additionally, a guide RNA which comprises labeled isotopes, such as one or more of15N,13C,14C, Deuterium, or32P, or other atoms used as tracers, is a modified guide RNA. Either independently or additionally, a guide RNA which comprises modifications made to the sugar group is a chemically modified RNA. Sugar group modifications can include but are not limited to 2’-O-methyl, 2’-deoxy, 2 ’-methoxy ethyl, 2’fluoro, 2’-amino, a sugar in L form, and 4’-thioribosyl.

[0179] In certain embodiments, chemical modifications protect the guide RNA from nucleases. In certain embodiments, this modification aids in the stability of the RNA molecules, where the half-life of the chemically modified RNA molecule is altered from the unmodified form. In certain embodiments, the chemically modified guide RNA maintains its functionality, which includes guide RNA binding to a Cas protein. In some embodiments, this maintained functionality of the gRNA includes binding a target polynucleotide. In some embodiments, the maintained functionality of the guide RNA includes binding both a Cas protein and a polynucleotide in complex. In some embodiments, the chemical modifications on the guide RNA are used to distinguish the sequences from the nascent sequences present in the experimental plant. In certain embodiments, the chemical modifications alter the prevalence of off-target cleavage events, where “off-target” is defined as a site in the target genome that is different from the site at which the guide RNA was designed to induce a cleavage event.

[0180] In some embodiments, the guide RNA and the Cas nuclease form a complex and introduce a single- or double-stranded break in the sequence of the gene of interest. In some embodiments, the guide RNA is directed to a phytoene desaturase (PDS) gene. In some embodiments, the guide RNA is directed to a gene contributing to an agronomic trait of interest.61MF-365821254Attorney Docket No.: 16536-20022.40In some embodiments, the gRNA is directed to a regulatory or coding sequence. In some embodiments, the regulatory or coding sequence contributes to a trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf size.

[0181] Chemical modifications to guide RNAs are known in the art, for example in U.S. Patent No. 10,337,001, and Ryan et al. 2018, Nucleic Acids Res. 46(20): 792-803.

[0182] In some embodiments the guide RNA further comprises (a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide RNA; or (b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide RNA; or (c) both (a) and (b); wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar. In some embodiments, each of the one or more modified nucleotides is independently selected from the group consisting of a 2'-O-methyl nucleotide, a 2'-0-methyl-3'-phosphorothioate nucleotide, a 2'-O-methyl-3'-phosphonoacetate nucleotide, and a 2'-0-methyl-3'-phosphonothioacetate nucleotide. In some embodiments, the one or more modified nucleotide comprises a modified internucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group.

[0183] In some embodiments, the nucleic acid encoding the guide RNA is linked to a promoter. In some embodiments, the promoter is an RNA polymerase II promoter or an RNA polymerase III promoter. In some embodiments, the RNA polymerase II promoter or RNA polymerase III promoter is endogenous to the species of the rootstock.

[0184] In some embodiments, a single guide RNA is provided to the plant. In other embodiments, multiple guide RNAs are provided to the plant. In some embodiments, the multiple guide RNAs are provided in a CRISPR array. In some embodiments, the two or more guide RNAs are encoded by a single precursor RNA. For the purposes of gene editing, CRISPR arrays can be designed to contain one or multiple guide RNAs designed to target a DNA sequence for editing, where the guide RNA includes at least one spacer sequence that corresponds to a specific locus of about equivalent length in the target DNA; see, for example, Cong et al. Science, 2013, 339: 819-823; Ran et al. Nature Protocols, 2013, 8: 2281-2308. In some embodiments, the CRISPR array comprises more than one spacer sequence. In some embodiments, the CRISPR array comprises more than one distinct spacer sequences. In some embodiments, the CRISPR array comprises more than one distinct spacer sequences designed 62MF-365821254Attorney Docket No.: 16536-20022.40to target the same genomic locus. In some embodiments, the CRISPR array comprises more than one distinct spacer sequences designed to target more than one distinct genomic loci. In some embodiments, the multiple guide RNAs are provided in a polycistronic system, wherein the multiple guide RNAs are linked to a single promoter. In other embodiments, the multiple guide RNAs are operable linked to multiple promoters. In some embodiments, the multiple guide RNAs are linked to multiple copies of the same promoter. In some embodiments, the multiple guide RNAs are linked to different promoters. In some embodiments, the multiple guide RNAs target the same genomic locus. In other embodiments, the multiple guide RNAs target multiple genomic loci. In some embodiments, the multiple guide RNAs are provided in a CRISPR array, wherein the CRISPR array is linked to a single engineered phloem mobility element. In some embodiments, the method comprises applying two or more, three or more, four or more, or five or more guide RNAs. In some embodiments, the two or more, three or more, four or more, or five or more guide RNAs are each linked to an engineered phloem mobility element. In some embodiments, the multiple guide RNAs are provided in a polycistronic system, wherein the multiple guide RNAs are linked to a single engineered phloem mobility element. In other embodiments, the multiple guide RNAs are operable linked to multiple engineered phloem mobility elements. In some embodiments, the multiple guide RNAs are linked to multiple copies of the same engineered phloem mobility element. In some embodiments, the multiple guide RNAs are linked to different engineered phloem mobility elements.

[0185] In some embodiments, delivery of the guide RNA comprises spraying a composition comprising the guide RNA onto the leaves, shoot, stem, and / or meristem. In some embodiments, the composition comprising the guide RNA comprises a surfactant. In some embodiments, the composition comprising the guide RNA comprises glass beads coated with the guide RNA.

[0186] In some embodiments, delivery of the guide RNA comprises rubbing a composition comprising the guide RNA onto the leaves, shoot, stem, and / or meristem.

[0187] In some embodiments, delivery of the guide RNA comprises infecting the plant with a viral vector. In some embodiments, the guide RNA is linked to an engineered phloem mobility element.

[0188] In some embodiments, delivery of the guide RNA comprises infecting the plant with an Agrobacterium vector. In some embodiments, the Agrobacterium vector comprises an engineered phloem mobility element.63MF-365821254Attorney Docket No.: 16536-20022.40

[0189] In some embodiments, delivery of the guide RNA comprises injecting a composition comprising the guide RNA into the stem. In some embodiments, the guide RNA is linked to an engineered phloem mobility element.

[0190] In some embodiments, delivery of the guide RNA comprises leaf infiltration of a composition comprising the guide RNA into the leaf. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. In some embodiments, the leaf infiltration comprises forced infiltration using a needle-less syringe or vacuum pump.

[0191] In some embodiments, the guide RNA is delivered to the plant root by incubating the root with a composition comprising the guide RNA. In some embodiments, the guide RNA is linked to an engineered phloem mobility element.

[0192] In some embodiments, the guide RNA is delivered to the plant root by an Agrobacterium rhizogenes transformation. In some embodiments, the guide RNA is linked to an engineered phloem mobility element. In some embodiments, the Agrobacterium rhizogenes transformation produces transgenic hairy roots.

[0193] In some embodiments, the guide RNA is delivered to the plant root by injecting a composition comprising the guide RNA into the root. In some embodiments, the guide RNA is linked to an engineered phloem mobility element.

[0194] In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor, optionally, wherein the nuclease inhibitor is an RNase inhibitor.

[0195] In some embodiments, the composition comprising the guide RNA comprises a nuclease inhibitor. In some embodiments, the nuclease inhibitor comprises an RNase inhibitor.

[0196] In some embodiments, application comprises biolistic transformation of nucleic acid encoding the guide RNA into the leaf, shoot, shoot, stem, and / or meristem. In some embodiments, the biolistic transformation comprises transformation of circular DNA encoding the guide RNA.I. Donor Templates

[0197] In certain embodiments, a donor DNA template is linked to an engineered phloem mobility element and provided as cargo in addition to the CRISPR Cas nuclease and the at least one gRNA, in order to effect incorporation of a DNA sequence from the donor DNA template at the target editing site in the plant genome by a mechanism such as, but not limited to, homology dependent repair (HDR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or transgene integration. In some embodiments, the64MF-365821254Attorney Docket No.: 16536-20022.40incorporation of a DNA sequence from the donor DNA template results in an insertion, deletion, and / or substitution in the DNA sequence at the target editing site in the plant genome.

[0198] In some embodiments, the virus-mediated delivery further comprises delivering a donor template DNA to the plant, optionally wherein the donor template DNA is delivered by infecting the plant with a viral vector that infects the meristem cell.

[0199] In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in an insertion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a deletion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a substitution in the DNA sequence at the target editing site in the plant genome. Donor DNA template molecules used in the methods provided herein include DNA molecules comprising, from 5’ to 3’, a first homology arm, a replacement DNA, and a second homology arm, wherein the homology arms contain sequences that are partially or completely homologous to genomic DNA (gDNA) sequences flanking a target site in the genomic DNA. In some embodiments, the target editing site in the genomic DNA overlaps the site targeted by the gRNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of one or more DNA base pairs relative to the target gDNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of one or more DNA base pairs relative to the site targeted by the gRNA. In one embodiment, the donor DNA template molecule is double- stranded and perfectly base-paired through all or most of its length. In another embodiment, the donor DNA template molecule is double- stranded and includes one or more non-terminal mismatches or non-terminal unpaired nucleotides within the otherwise double- stranded duplex. In an embodiment, the donor DNA template molecule that is integrated at the site of at least one double-strand break (DSB) includes between 2-20 nucleotides in one (if single- stranded) or in both strands (if double-stranded), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands, each of which can be base-paired to a nucleotide on the opposite strand of the targeted integration site (in the case of a perfectly base-paired double- stranded polynucleotide molecule). Such donor DNA templates can be integrated in genomic DNA containing blunt and / or staggered double stranded DNA breaks by a mechanism such as, but not limited to, homology dependent repair (HDR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or transgene integration. In certain embodiments, a donor DNA template homology arm can be about 20, 50, 100, 200, 400, or 600 to about 800, or 1000 base 65MF-365821254Attorney Docket No.: 16536-20022.40pairs in length. In certain embodiments, a donor DNA template molecule can be delivered to a plant cell in a circular (e.g., a plasmid or a viral vector including a geminivirus vector or comovirus vector) or a linear DNA molecule. In certain embodiments, a circular or linear DNA molecule that is used can comprise a modified donor DNA template molecule comprising, from 5’ to 3’, a first copy of the target gRNA site sequence, the first homology arm, the replacement DNA, the second homology arm, and a second copy of the target gRNA site sequence. In other embodiments, DNA templates suitable for NHEJ insertion will lack homology arms that are partially or completely homologous to gDNA sequences flanking a target site-specific nuclease cleavage site in the gDNA. Compositions comprising the donor templates can be delivered to the plant and / or meristem cells of the plant by viral delivery, and other methods of delivery, such as but not limiting to, Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, and / or by use of cell-penetrating peptides. The donor template may be delivered by plasmid. The donor DNA templates may be present transiently in the cell or it could be introduced via a viral replicon (e.g., a geminivirus replicon). Geminivirus DNA replicons suitable for delivery of donor DNA templates to plants include a Beet Yellow Dwarf Virus replicon (Baltes, N.J. et al. Plant Cell vol. 26, 1 (2014): 151-63.). In some embodiments, the method further comprises delivering a donor template DNA to the plant by virus-mediated delivery. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the plant. In some embodiments, the sequence is incorporated into the genome of the plant at the genomic target. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the scion. In some embodiments, the donor template DNA is delivered to the scion using the same viral vector as the gRNA. In some embodiments, the donor template DNA is delivered to the plant using the same viral vector as the gRNA. In some embodiments, the donor template DNA is delivered to the scion using a different viral vector than is used to deliver the gRNA. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the scion at the locus targeted by the gRNA. In some embodiments, the donor template DNA confers a desired trait. In some embodiments, the donor template comprises an endogenous sequence. In other embodiments, the donor template comprises an exogenous sequence. Donor templates can be utilized in VIGE alongside systems such as retron systems. In some embodiments, the method further comprises delivering a donor template DNA to the plant by virus-mediated delivery. In some embodiments, the donor template is delivered by infecting the plant with a vector, optionally an Agrobacterium vector. In some embodiments, a sequence from the donor 66MF-365821254Attorney Docket No.: 16536-20022.40template DNA is incorporated into the genome of the plant. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the plant at the gene of interest. In some embodiments, the donor template is co-transfected with the guide RNA. In some embodiments, the donor template DNA is delivered to the plant using a different viral vector than the viral vector carrying the guide RNA. In some embodiments, the donor template DNA is delivered to the plant using a different vector than the vector carrying the guide RNA.

[0200] In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in an insertion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a deletion in the DNA sequence at the target editing site in the plant genome. In some embodiments, the incorporation of a DNA sequence from the donor DNA template results in a substitution in the DNA sequence at the target editing site in the plant genome. Donor DNA template molecules used in the methods provided herein include DNA molecules comprising, from 5’ to 3’, a first homology arm, a replacement DNA, and a second homology arm, wherein the homology arms contain sequences that are partially or completely homologous to genomic DNA (gDNA) sequences flanking a target site in the genomic DNA. In some embodiments, the target editing site in the genomic DNA overlaps the site targeted by the gRNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of one or more DNA base pairs relative to the target gDNA. In one embodiment, the donor DNA template molecule is double-stranded and perfectly base-paired through all or most of its length, with the possible exception of any unpaired nucleotides at either terminus or both termini. In another embodiment, the donor DNA template molecule is double-stranded and includes one or more non-terminal mismatches or non-terminal unpaired nucleotides within the otherwise double- stranded duplex. In an embodiment, the donor DNA template molecule that is integrated at the site of at least one double-strand break (DSB) includes between 2-20 nucleotides in one (if single- stranded) or in both strands (if double-stranded), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands, each of which can be base-paired to a nucleotide on the opposite strand of the targeted integration site (in the case of a perfectly base-paired double- stranded polynucleotide molecule). Such donor DNA templates can be integrated in genomic DNA containing blunt and / or staggered double stranded DNA breaks by a mechanism such as, but not limited to, homology dependent repair (HDR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or transgene integration. In certain embodiments, a donor DNA template homology arm can be about 20, 50, 100, 200, 400, or 600 to about 800, or 1000 base 67MF-365821254Attorney Docket No.: 16536-20022.40pairs in length. In certain embodiments, a donor DNA template molecule can be delivered to a plant cell in a circular (e.g., a plasmid or a viral vector including a geminivirus vector) or a linear DNA molecule. In certain embodiments, a circular or linear DNA molecule that is used can comprise a modified donor DNA template molecule comprising, from 5’ to 3’, a first copy of the target gRNA site sequence, the first homology arm, the replacement DNA, the second homology arm, and a second copy of the target gRNA site sequence. In other embodiments, DNA templates suitable for NHEJ insertion will lack homology arms that are partially or completely homologous to gDNA sequences flanking a target site-specific nuclease cleavage site in the gDNA. Compositions comprising the donor templates can be delivered to the plant and / or meristem cells of the plant by particle mediated delivery, and any other direct method of delivery, such as but not limiting to, Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, and / or by use of cell-penetrating peptides. The donor DNA templates may be present transiently in the cell or it could be introduced via a viral replicon (e.g., a geminivirus replicon). Geminivirus DNA replicons suitable for delivery of donor DNA templates to plants include a Beet Yellow Dwarf Virus replicon (Baltes, N.J. et al. Plant Cell vol. 26, 1 (2014): 151-63.). In some embodiments, the method further comprises delivering a donor template DNA to the plant by virus-mediated delivery. In some embodiments, a sequence from the donor template DNA is incorporated into the genome of the scion. In some embodiments, the donor template DNA is delivered to the scion using the same viral vector as the gRNA. In some embodiments, the donor template DNA is delivered to the scion using a different viral vector than is used to deliver the gRNA. In some embodiments, the sequence from the donor template DNA is incorporated into the genome of the scion at the locus targeted by the gRNA. In some embodiments, the donor template DNA confers a desired trait. In some embodiments, the donor template comprises an endogenous sequence. In other embodiments, the donor template comprises an exogenous sequence.

[0201] In some embodiments, a donor template is delivered to a meristem cell of a plant, wherein the donor template is an mRNA fused to an engineered phloem mobility element. In some embodiments, the donor template is delivered to a meristem cell for gene editing as a part of a retron reverse transcriptase system, (see, for example, W02025007020, which is hereby incorporated by reference in its entirety). In some embodiments, the retron reverse transcriptase is delivered separately to the meristem cell, optionally wherein the retron reverse transcriptase is delivered as an RNA cargo attached to an engineered phloem mobility element described herein.68MF-365821254Attorney Docket No.: 16536-20022.40J. Prime Editing

[0202] Desired DNA sequence modifications can be accomplished through the use of PRIME editing (Anzalone et al. Nature 2019, 576(7785): 149-157). In some embodiments, prime editing uses (i) a Cas nickase, in some embodiments a Cas9 nickase, in other embodiments a Casl2 nickase, fused to a reverse transcriptase (nCas-RT), in some embodiments a M-MLV reverse transcriptase, and (ii) a prime editing Cas guide RNA (pegRNA) that both specifies the genome target site and has an extension that encodes the target edit within a template for the reverse transcriptase. In some embodiments, gRNA is a prime editing guide RNA (pegRNA). The binding of the pegRNA directs the Cas nickase to create a single-stranded break in the DNA at the nicking site. The extension of the pegRNA binds to the nicked DNA that has an exposed 3 ’-hydroxyl group, priming the reverse transcriptase to produce a DNA strand that is complementary to the extension of the pegRNA. This DNA strand will include the complement to any desired edits present in the provided pegRNA extension. Mismatch repair by the cell will then resolve the mismatch between the unedited parent strand and the edited product of the reverse transcriptase, thus introducing the desired edits into the genome. Prime editing systems may also include elements to inhibit mismatch repair, or to nick the unedited parent strand to increase editing efficiency. An engineered phloem mobility element can be linked to the pegRNA so as not to interfere with priming of the reverse transcriptase. In some embodiments of a viral vector system for use in plant editing, the system comprises a plant virus genome component; one or more pegRNA; and a Cas nuclease fused to a reverse transcriptase, wherein the pegRNA and / or the Cas nuclease fused to the reverse transcriptase is linked to an engineered phloem mobility element. In some embodiments, the donor template DNA is delivered to the plant using a different viral vector than the viral vector comprising the gRNA. In some embodiments, the donor template DNA confers a desired trait on the plant. In some embodiments, the donor template is co-transfected with the guide RNA. In some embodiments, the donor template DNA is delivered to the plant using a different viral vector than the viral vector carrying the guide RNA. In some embodiments, the donor template comprises an exogenous sequence. In some embodiments, the donor template comprises an endogenous sequence. In some embodiments, the donor template is linked to an engineered phloem mobility element.

[0203] Desired DNA sequence modifications can be accomplished through the use of PRIME editing (Anzalone et al. Nature 2019, 576(7785): 149-157). In some embodiments, prime editing uses (i) a Cas nickase, in some embodiments a Cas9 nickase, in other embodiments a69MF-365821254Attorney Docket No.: 16536-20022.40Casl2 nickase, fused to a reverse transcriptase (nCas-RT), in some embodiments a M-MLV reverse transcriptase, and (ii) a prime editing Cas guide RNA (pegRNA) that both specifies the genome target site and has an extension that encodes the target edit within a template for the reverse transcriptase . The binding of the pegRNA directs the Cas nickase to create a singlestranded break in the DNA at the nicking site. The extension of the pegRNA binds to the nicked DNA that has an exposed 3 ’-hydroxyl group, priming the reverse transcriptase to produce a DNA strand that is complementary to the extension of the pegRNA. This DNA strand will include the complement to any desired edits present in the provided pegRNA extension. Mismatch repair by the cell will then resolve the mismatch between the unedited parent strand and the edited product of the reverse transcriptase, thus introducing the desired edits into the genome. Prime editing systems may also include elements to inhibit mismatch repair, or to nick the unedited parent strand to increase editing efficiency. An engineered phloem mobility element can be linked to the pegRNA so as not to interfere with priming of the reverse transcriptase.

[0204] In some embodiments, prime editing can also be accomplished with Cas nucleases in place of Cas nickases (Adikusuma et al. Nucleic Acids Res. 2021, 49(18): 10785-10795). In some embodiments, prime editing uses (i) a Cas nuclease, in some embodiments a Cas9 nuclease, in other embodiments a Cas 12 nuclease, fused to a reverse transcriptase (Cas-RT), in some embodiments a M-MLV reverse transcriptase, and (ii) a prime editing Cas guide RNA (pegRNA) that both specifies the genome target site and has an extension that encodes the target edit within a template for the reverse transcriptase. In some embodiments, the binding of the pegRNA directs the Cas nuclease to create a double-stranded break in the DNA at the target site. The extension of the pegRNA binds to the cut DNA that has an exposed 3 ’-hydroxyl group, priming the reverse transcriptase to produce a DNA strand that is complementary to the extension of the pegRNA. This DNA strand will include the complement to any desired edits present in the provided pegRNA extension. Mismatch repair by the cell will then resolve the mismatch between the unedited parent strand and the edited product of the reverse transcriptase, thus introducing the desired edits into the genome. Prime editing systems may also include elements to inhibit mismatch repair, or to nick the unedited parent strand to increase editing efficiency. An engineered phloem mobility element can be linked to the pegRNA so as not to interfere with priming of the reverse transcriptase.

[0205] Prime editing makes precise DNA sequence modifications rather than random insertions, deletions, and substitutions (Indels), thus increasing the probability of obtaining the desired effect. Prime editing may be used to introduce any single base pair substitution as well 70MF-365821254Attorney Docket No.: 16536-20022.40as small deletion or insertions. Deletions of up to 80 base pairs have been produced using prime editing with a single pegRNA in human cells, and insertions of up to 40 base pairs (Anzalone et al. Nature 2019, 576: 149-157). Dual pegRNA systems are also known in the art (Choi et al. Nat Biotechnol 2021, 40(2): 218-226; Lin et al. Nature Biotechnology 2021, 39(8): 923-927) and can be used to generate precise large deletions, or to improve editing efficiency for small insertions, deletions, or substitutions. Additionally, dual pegRNA systems where the extension of the pegRNAs are not complementary to the endogenous locus, but are complementary to one another, can be used to replace endogenous sequence and / or mediate larger insertions (Anzalone et al. Nat Biotechnol 2022, 40(5): 731-740).

[0206] In some embodiments, the Cas nuclease is associated with a reverse transcriptase. In some embodiments, the Cas nuclease is fused to the reverse transcriptase. In some embodiments, the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target. In some embodiments, the Cas nuclease is a Cas nickase. In some embodiments, the Cas nickase is a Cas9 nickase or a Cas 12 nickase. In some embodiments, the Cas nickase comprises mutation in one or more nuclease active sites.K. Delivery to the Meristem

[0207] In some embodiments, the methods provided herein involve transport of one or more RNA cargos to the meristem. Engineered phloem mobility elements travel through the plant, typically but not limited to via the phloem, and are taken up into meristematic tissues. Examples of engineered phloem mobility elements and their secondary structures are described above in Section II. In some embodiments of the invention, a first RNA cargo is linked to an engineered phloem mobility element, and a second RNA cargo is linked to a second phloem mobility element. In some embodiments, the second phloem mobility element is a second engineered phloem mobility element as described in the above sections. In some embodiments, the second phloem mobility element is a meristem transport segment, which travels through the plant, typically but not limited to via the phloem, and is taken up into meristematic tissues. The examples below are sequences from individual species, which sometimes work across species. For example, Arabidopsis FT-based vectors work in Nicotiana benthamiana and Arabidopsis. Vectors can also be designed based on alternative sequences, which can be based either on the species subject to genomic editing or based on a different species, sometimes a related species, sometimes a closely related species.

[0208] While the transport segment is based on a plant-transported RNA, its actual sequence may be a fragment determined by characterizing a deletion series to make a smaller sequence 71MF-365821254Attorney Docket No.: 16536-20022.40retaining the desired transport (phloem mobility and / or meristem cell translocation) capabilities. The initiator methionine codon or translation initiation codon of the base sequence may also be mutated in some cases.

[0209] The phloem mobility competence potential can be determined for any candidate engineered phloem mobility element. A side-by-side comparison with a known engineered phloem mobility element, such as the constructs previously tested in planta in Example 2, as a positive control is useful. As such, a number of configurations can be used. One approach is to link candidate sequences to guide sequences of characterized editing potential for a species of interest. RNA sequences can be introduced into the phloem of an individual plant that expresses or translates at least in the meristem a nuclease capable of associating with the guide sequence and producing the intended genomic alteration. The RNA sequences can be expressed in vitro and introduced into the phloem as substantially purified molecules. Producing inoculum and / or thoughtful selection and preparation of recipient plant tissue can greatly increase the levels of success for infecting a plant with a viral vector or composition comprising a recombinant plant virus. For example, a concentrated solution of RNA molecules of interest can be applied to a mechanically injured plant tissue, such as a cut or abraded leaf, stem, meristem-associated tissue, or any vegetative tissue. RNAs can be coated on particles, such as micro or nano-scale particles such as gold or tungsten, for biolistic delivery. Alternatively, the guide RNA sequences can be incorporated into RNA viruses introduced in the plants (Jackson et al. Front. Plant Sci. 2012, 3: 127; Ali et al. Mol. Plant 2015, 8: 1288-1291; Cody et al. Plant Physiol.2017, 175: 23-35; Ali et al. Virus Res. 2018, 244: 333-337; Gao et al. New Phytol. 2019, 223: 2120-2133) or the MTC can be assayed by introducing RNAs by grafting, i.e., the RNA molecules can be expressed in the rootstock of a grafted plant, and their effect observed in the scion (Zhang et al. Plant Cell, 2016, 28: 1237-1249; Huang et al. Plant Physiol. 2018, 178:783-794). Candidate engineered phloem mobility elements can be assayed for longer and / or more complex RNA molecules, or mixtures of RNA molecules, that comprise not only guide or processable guide regions, but also nuclease-encoding sequences. Ideal viral vector candidates can be assayed for longer and / or more complex RNA molecules, or mixtures of RNA molecules, that comprise not only guide or processable guide regions, but also nuclease-encoding sequences. A clear readout of the phloem transport competence potential is detection of the expected genomic alterations in progeny plants, which can be done by sequencing of the target genomic region, or even by whole genome sequencing. A clear readout of successful delivery to the meristem and modification of the meristem is detection of the expected genomic 72MF-365821254Attorney Docket No.: 16536-20022.40alterations in progeny plants, which can be done by sequencing of the target genomic region, or even by whole genome sequencing. A clear readout of successful delivery to the meristem cell and modification of the meristem cell is detection of the expected genomic alterations in progeny plants, which can be done by sequencing of the target genomic region, or even by whole genome sequencing, but alternative readouts can be designed that may be more convenient in some cases. For example, the guide sequences may be directed to disrupt or repair a reporter gene, such as a transgene encoding a fluorescent polypeptide. The expected genetic changes can then be evaluated in the treated plants by measuring changes in the reporter. Another convenient genomic alteration target in many species is phytoene desaturase (PDS), with the albino phenotype as a result of photobleaching of the mutant serving as a readout (see, for example, Kumagai et al. PNAS 1995, 92(5): 1679-1683; Xie et al. PNAS 2015, 112(11): 3570-3575).

[0210] In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas nuclease and / or 3’ of the nucleic acid encoding the guide RNA. In some embodiments, the engineered phloem mobility element is located 5’ of the nucleic acid encoding the Cas nuclease and / or 5’ of the nucleic acid encoding the guide RNA. In some embodiments, the engineered phloem mobility element is located 3’ of the nucleic acid encoding the Cas nuclease and / or 3’ of the nucleic acid encoding the guide RNA.

[0211] In some embodiments, the plant further comprises nucleic acid encoding a detectable marker linked to a phloem mobility element, such as an engineered phloem mobility element as described herein, or a meristem transport segment (MTS).L. Genome Modifications

[0212] In some embodiments, the reagents and methods described provide a relatively easy and convenient solution for producing plants with altered genomes, i.e., individuals with designed DNA sequence modifications (e.g., Indels or epigenetic alterations). In some embodiments, the methods provided herein can be applied to provide RNA cargo comprising genome editing reagents to a plant, and thereby edit one or more genomic regions selected independently from the group consisting of a gene, an array of tandemly duplicated genes, a multigene family, an enhancer, a suppressor, a promoter, a termination sequence, a splice acceptor sequence, a splice donor sequence, an intron, an exon, an siRNA, a sequence encoding a non-coding RNA, a microRNA, a transgene, and a quantitative trait locus (QTL). In some embodiments, the edit results in the insertion or deletion of nucleotides at or near the target73MF-365821254Attorney Docket No.: 16536-20022.40sequence. In some embodiments, the edit results in an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 nucleotides at or near the target sequence. In some embodiments, the edit results in a nucleotide substitution at or near the target sequence. In some embodiments, the edit results in a substitution of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides at or near the target sequence. In most embodiments, the methods and systems rely on DNA or RNA molecules produced with established molecular biology techniques. The DNA or RNA molecules, which comprise genome-editing reagents, are then introduced into a plant and taken up into meristematic cells. The meristematic cell genomes are thus altered, and the DNA sequence modifications (e.g., Indels or epigenetic alterations) are carried into germline cells and subsequent generations.

[0213] Very often, mutated seeds from plants edited with the reagents and methods described here are collected for phenotypic characterization. In some cases, pollen from edited plants is used in crosses with other individuals, or mutated individuals are pollinated with pollen of unedited plants or wildtype plants. In some embodiments, the method of editing a genomic target in a meristem cell of a plant comprises virus-mediated delivery, wherein the virus-mediated delivery comprises infecting the plant with an Agrobacterium vector carrying a guide RNA (gRNA) directed to the genomic target in the meristem cell of the plant. In some embodiments, the method further comprises screening the plant for viral infection, said screening comprising a visual assessment of the plant for a desired phenotype. In some embodiments, the method further comprises screening the plant for successful genome modification, said screening comprising visually assessing the plant for desired phenotype. In some embodiments, said screening further comprises sequencing of cells produced by the meristem after delivery of the Agrobacterium vector. In some embodiments, the method further comprises screening the progeny of the plant for successful genome modification, said screening comprising visually assessing a plant that grows from the seed for desired phenotype, and / or sequencing of cells.

[0214] The embodiments’ described methods and reagents can have many advantages over other known solutions. The techniques presented generally bypass callus induction or tissue culture that are necessary for alternative or widely practiced genome editing procedures, thus speeding up (i.e., accelerating) and lowering or reducing the cost of the process of producing plants with targeted DNA sequence modifications. Epigenetic resetting (i.e., interference) is also eliminated. The editing can be performed in individuals of an elite genetic background, making lengthy backcrossing schemes unnecessary.74MF-365821254Attorney Docket No.: 16536-20022.40

[0215] Plants comprising the RNA molecules that comprise the RNA cargo, provided to the plant via linked transport with an engineered phloem mobility element are provided herein. In some embodiments, the RNA cargo comprises one or more genome editing reagents. In some embodiments plants comprising the RNA molecules that comprise a Cas nuclease and / or guide RNA(s) linked to an engineered phloem mobility element are also provided herein. Plants comprising the RNA molecules that comprise a Cas nuclease and / or guide RNA(s), wherein the Cas nuclease and / or guide RNAs are linked to engineered phloem mobility elements sequences are also provided herein. In certain embodiments, such RNA molecules will be present at detectable concentrations in the plants for only a certain period of time following a stimulus. For example, the concentrations of RNA molecules comprising guide RNAs separated by processing elements comprising direct repeats (DR, i.e., pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA)) which are capable of being processed (i.e., cleaved) by an RNA-guided nuclease are expected to decrease over time when the RNA-guided nuclease is also present in the plant. The concentrations of RNA molecules comprising guide RNAs separated by processing elements comprising direct repeats which are capable of being processed by an RNA-guided nuclease are also expected to be decreased in tissues where the RNA-guided nuclease is located. Nonetheless, the unprocessed RNA molecules can be detected by a variety of techniques that include reverse transcription polymerase chain reaction (RT-PCR) assays where oligonucleotide primers and optionally detection probes which specifically amplify and detect the unprocessed RNA molecule comprising the Cas nuclease and / or guide RNA(s) that are linked to engineered phloem mobility elements are used. The unprocessed RNA molecules can also be detected by a variety of techniques that include reverse transcription polymerase chain reaction (RT-PCR) assays where oligonucleotide primers and optionally detection probes which specifically amplify and detect the unprocessed RNA molecule comprising the viral vector, Cas nuclease, and / or guide RNA(s) are used. Such plants can comprise any of the RNA molecules or combinations of RNA molecules present in the compositions provided herein that are used to contact the plants. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in meristem tissue of the plant. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in meristem cells of the plant. In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that further comprises a linked engineered phloem mobility element. In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises a viral vector. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is linked to promoters that include a root- 75MF-365821254Attorney Docket No.: 16536-20022.40preferred or root-specific promoter which is active in root cells. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is linked to constitutively active promoters. DNA encoding the RNA-guided nuclease can be provided in a transgene that is stably integrated in the genome of the plant, in DNA that is not integrated into the plant genome, or in DNA provided in a viral vector (e.g., a geminivirus replicon). Geminivirus DNA replicons suitable for delivery of DNA molecules encoding an RNA-guided nuclease to plants include a Beet Yellow Dwarf Virus replicon (Baltes et al. Plant Cell 2014, 26(1): 151-63; doi: 10[dot] 1105 / tpc[dot] 113 [dot] 119792).

[0216] Nonetheless, the unprocessed RNA molecules can be detected by a variety of techniques that include reverse transcription polymerase chain reaction (RT-PCR) assays where oligonucleotide primers and optionally detection probes which specifically amplify and detect the unprocessed RNA molecule comprising the viral vector, Cas nuclease and / or guide RNA(s) are used. Such plants can comprise any of the RNA molecules or combinations of RNA molecules present in the compositions provided herein that are used to contact the plants. In certain embodiments, an active form of the RNA guided nuclease is predominantly localized in meristem cells of the plant. In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises a linked engineered phloem mobility element. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is linked to promoters that include a root-preferred or root-specific promoter which is active in root cells. In certain embodiments, the RNA-guided nuclease can be encoded by DNA that is linked to constitutively active promoters. DNA encoding the RNA-guided nuclease can be provided in a transgene that is stably integrated in the genome of the plant, in DNA that is not integrated into the plant genome, or in DNA provided in a viral vector (e.g., a geminivirus replicon). Geminivirus DNA replicons suitable for delivery of DNA molecules encoding an RNA-guided nuclease to plants include a Beet Yellow Dwarf Virus replicon (Baltes et al. Plant Cell 2014, 26(1): 151-63; doi: 10[dot] 1105 / tpc[dot] 113[dot] 119792). In certain embodiments, the RNA-guided nuclease can be encoded by an RNA molecule that optionally further comprises a viral vector.

[0217] Plants comprising the RNA cargo linked to engineered phloem mobility elements are also provided herein. In some embodiments, plants comprising the RNA molecules that comprise a Cas nuclease and / or guide RNA(s) that are linked to engineered phloem mobility elements are also provided herein. Also provided herein are plants that comprise the RNA molecules that comprise a Cas nuclease and / or guide RNA(s) that are linked to engineered phloem mobility elements. In certain embodiments, such RNA molecules will be present at 76MF-365821254Attorney Docket No.: 16536-20022.40detectable concentrations in the plants for only a certain period of time following a stimulus. In some embodiments, viral vectors delivered as DNA are then processed into RNA by transcription. In certain embodiments, an engineered phloem mobility element is linked to a CRISPR Cas system comprising a plurality of guide RNAs (e.g., 2, 3, 4, or more guide RNAs) separated by processing elements to provide for gene editing at a plurality of genomic locations targeted by each guide RNA. In certain embodiments, a viral vector comprises a linked CRISPR Cas system comprising a plurality of guide RNAs (e.g., 2, 3, 4, or more guide RNAs) separated by processing elements to provide for gene editing at a plurality of genomic locations targeted by each guide RNA. In certain embodiments, the plurality of guide RNAs are separated by processing elements comprising direct repeats (DR; i.e., pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA)) which are capable of being processed (i.e., cleaved) by an RNA-guided nuclease. Examples of such DRs include the Casl2a DR (e.g., SEQ ID NO: 54 or 56) which can be cleaved by a Cas 12a guided nuclease (e.g., SEQ ID NO: 53 or 55, respectively). Cleavage of RNAs comprising Casl2a DRs by Casl2a has been described (Fonfara et al. Nature 2016, 532: 517-521, doi[dot]org / 10[dot]1038 / naturel7945); US20160208243; WO 2017 / 189308). Other examples of such DRs include the Casl2j DRs (e.g., SEQ ID NO: 58, 60, or 62) which can be cleaved by a Casl2j guided nuclease (e.g., SEQ ID NO: 57, 59, or 61, respectively). In such embodiments, the crRNA portion of the DR can remain as a part of the gRNA after processing and can be recognized by the RNA guided nuclease to provide for editing of genomic DNA recognized via hybridization of the gRNA to the targeted genomic site.

[0218] In some embodiments, the meristem is part of a plant scion grafted onto a rootstock. In other embodiments, the meristem is part of a non-grafted plant.M. Targets of Genomic Modification

[0219] In some embodiments, embodiments of the engineered phloem mobility elements, compositions, engineered systems, and methods disclosed herein are useful in increasing transport of genome editing reagents, and thereby increasing the rate of editing or effecting a sequence-specific modification of a target DNA sequence or target gene in a DNA molecule, a chromosome, or a genome. Embodiments of the engineered phloem mobility elements, T-DNA vector system and methods of use disclosed herein are useful in editing or effecting a sequencespecific modification of a target DNA sequence or target gene in a DNA molecule, a chromosome, or a genome. In embodiments, the target sequence or target gene includes coding sequence (DNA encoding a polypeptide, such as a structural protein or an enzyme), non-coding 77MF-365821254Attorney Docket No.: 16536-20022.40sequence, or both coding and non-coding sequence. Embodiments of the polynucleotides, compositions, engineered systems, Agrobacterium vector systems, other T-DNA vector systems, and methods disclosed herein are useful in increasing transport of genome editing reagents within a plant, and therefore editing or effecting a sequence-specific modification of a target DNA sequence or target gene in a DNA molecule, a chromosome, or a genome. In embodiments, the target sequence or target gene includes coding sequence (DNA encoding a polypeptide, such as a structural protein or an enzyme), non-coding sequence, or both coding and non-coding sequence.N. Identification of Targets

[0220] There are numerous plant-endogenous targets (i.e., DNA sequence targets) for genome editing. The methods presented here can be applied to transport genome editing reagents to a plant cell, and thereby edit one or more genomic regions selected independently from the group consisting of a gene, an array of tandemly duplicated genes, a multigene family, an enhancer, a suppressor, a transcription factor binding site, a protein binding site, a promoter, a termination sequence, a splice acceptor sequence, a splice donor sequence, an intron, an exon, an siRNA, a sequence encoding a non-coding RNA, a microRNA, a transgene, an intergenic region, a genic region, a heterochromatic region, a euchromatic region, a region of methylated DNA, and a quantitative trait locus (QTL).

[0221] The method of the present invention may be used to provide RNA cargo comprising genome editing reagents to thereby introduce edits to affect any phenotype, quality, or trait of the organism. For instance, the methods herein may be used to introduce edits to the genome that affect yield, overall fitness, biomass, photosynthetic efficiency, nutrient use efficiency, heat tolerance, drought tolerance, herbicide tolerance, or disease resistance of a plant. In some embodiments, the viral vector carries at least one guide RNA, wherein the guide RNA is linked to an engineered phloem mobility element. In some embodiments, the T-DNA vector carries at least one guide RNA, wherein the guide RNA is linked to an engineered phloem mobility element. In some embodiments, the Agrobacterium vector carries at least one guide RNA, wherein the guide RNA is linked to an engineered phloem mobility element. In some embodiments, the guide RNA is directed to a regulatory or coding sequence contributing to trait selected from the group consisting of: photosynthetic ability or efficiency; yield or fertility; seed number; disease or pest resistance; herbicide or pesticide tolerance; abiotic stressor tolerance; fruit morphology; fruit nutrition; fruit ripening; number of seeds per pod; and leaf78MF-365821254Attorney Docket No.: 16536-20022.40size. In some embodiments, the guide RNA comprises a spacer that is complementary to a target sequence. In some embodiments, the target sequence is located within a target gene or a target genomic region.

[0222] The methods presented here can be applied to a promoter bashing or fine-tuning approach, to create a range of phenotypes based on promoter alterations of a gene of a certain sequence or gene of interest (Rodriguez-Leal et al. Cell 2017, 171(2): 470-480). For example, a target gene may be selected that has a current, baseline level of expression in a target plant species. Guide RNAs may be produced that target different regions of the promoter of this target gene. Multiple lines of the elite germplasm may be generated containing distinct edits in the target gene promoter using the methods provided herein. For example, one line may have deleted a transcription factor binding site; a second line may have introduced a single base pair substitution in the transcription factor binding site; a third line may have introduced two base pair substitutions in the transcription factor binding site. The differentially edited promoters can be assessed for phenotype, including sub-organismal level phenotype such as RNA expression level, gene transcript splicing ratio, ribosomal occupancy, allele specific expression, metabolite abundance, protein modifications, micro RNA or small RNA abundance, protein abundance, or translational efficiency, and / or organismal level phenotype such as yield, overall fitness, biomass, photosynthetic efficiency, nutrient use efficiency, heat tolerance, drought tolerance, herbicide tolerance, disease resistance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content. In some embodiments, the edit results in increased transcription compared to the baseline level of expression in a target plant species. In some embodiments, the edit results in decreased transcription compared to the baseline level of expression in a target plant species. The optimal allele may be selected based on sub-organismal phenotype and / or organismal phenotype.

[0223] Any defective, deleterious, non-optimal, or underperforming allele found in elite germplasm can be edited to a non-deleterious or more optimal allele. In some embodiments, a target to be modified is a genetic variant that is known in the art to be deleterious. In some embodiments, a target to be modified is identified by a linkage study or an association study, such as a genome-wide association study (GWAS) or a transcriptome-wide association study (TWAS). In some embodiments, a target to be modified is identified through the use of statistical models, machine learning, or artificial intelligence. Deleterious genetic variants may be identified through analysis of factors including, but not limited to, evolutionary conservation 79MF-365821254Attorney Docket No.: 16536-20022.40(See e.g. Chun and Fay Genome Res 2009, 19: 1553-1561; Rodgers-Melnick et al. PNAS 2015, 112: 3823-3828), functional impact of amino acid change (See e.g. Ng et al. NAR 2003, 31: 3812-3814; Adzhubei et al. Nat Methods 2010, 7: 248-249), functional impact of protein conformation and / or stability (See e.g. Rosetta, a computational protein design platform from Cyrus Bio Inc.), adjacency to selective sweep regions (See e.g. Hufford et al. Nat Gen 2012, 44: 808-813), and outlier status of a sub-organismal level phenotype such as RNA expression level, gene transcript splicing ratio, ribosomal occupancy, allele specific expression, metabolite abundance, protein modifications, micro RNA or small RNA abundance, protein abundance, or translational efficiency (See e.g. Zhao et al. AJHG 2016, 98: 299-309).

[0224] Editing of coding sequences can be made using the methods disclosed herein to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application Ser. No. 08 / 740,682, filed Nov. 1, 1996, and WO 98 / 20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley et al. Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Ill.) 1989, pp. 497-502; herein incorporated by reference); com (Pedersen et al. J. Biol. Chem. 1986, 261: 6279; Kirihara et al. Gene 1988, 71: 359; both of which are herein incorporated by reference); and rice (Musumura et al. Plant Mol. Biol. 1989, 12: 123, herein incorporated by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

[0225] The methods disclosed herein can be used to modify herbicide resistance traits including genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing DNA sequence modifications leading to such resistance, in particular the S4 and / or Hra modifications), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene); glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03 / 092360); or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron. Additional herbicide resistance traits are described for example in U.S. Patent Application 2016 / 0208243, herein incorporated by reference.80MF-365821254Attorney Docket No.: 16536-20022.40

[0226] Sterility genes can also be modified and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development. Additional sterility traits are described, for example, in U.S. Patent Application 2016 / 0208243, herein incorporated by reference.

[0227] Genome editing can also be used to make haploid inducer lines as disclosed in WO2018086623 and US20190292553.

[0228] The quality of grain can be altered by modifying genes encoding traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0229] Commercial traits can also be altered by modifying a gene or that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of modified plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as beta-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. J. Bacteriol 1988, 170: 5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).

[0230] Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.

[0231] The methods disclosed herein can also be used for modification of native plant gene expression to achieve desirable plant traits, such as an agronomically desirable trait. Such traits include, for example, disease resistance, herbicide tolerance, drought tolerance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content. Genes capable of conferring these desirable traits are disclosed in U.S. Patent Application 2016 / 0208243, herein incorporated by reference.

[0232] In some embodiments, edits generated by the methods provided herein are evaluated for changes in phenotype on a sub-organismal level, including evaluation of RNA expression level, gene transcript splicing ratio, ribosomal occupancy, allele specific expression, metabolite 81MF-365821254Attorney Docket No.: 16536-20022.40abundance, protein modifications, micro RNA or small RNA abundance, protein abundance, and / or translational efficiency. In some embodiments, edits generated by the methods provided herein are evaluated for changes in phenotype on an organismal level, including yield, overall fitness, biomass, photosynthetic efficiency, nutrient use efficiency, heat tolerance, drought tolerance, herbicide tolerance, disease resistance, salt tolerance, insect resistance, resistance against parasitic weeds, improved plant nutritional value, improved forage digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased storage life of plants or plant parts, reduced allergen production, and increased or decreased lignin content. The optimal allele and / or edits may be selected based on sub-organismal phenotype and / or organismal phenotype.O. Plants

[0233] The present disclosure may be used for increasing transport of RNA cargo within any plant species. In some embodiments, the plant species is selected from any species of soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana, cotton, or wheat. In some embodiments, the present disclosure may be used for increasing transport of RNA cargo within a monocot. In some embodiments, the present disclosure may be used for increasing transport of RNA cargo within a dicot.

[0234] In some embodiments, the plant is transfected with Agrobacterium-mediated transformation, thereby providing the RNA cargo linked to the engineered phloem mobility element to the plant. In some embodiments, the RNA cargo comprises one or more genome editing reagents and the method comprises editing a genomic target in the plant, for example, in a meristem cell of the plant. In some embodiments, a guide RNA linked to an engineered phloem mobility element is provided to the plant by a hairy root system, and an RNA encoding a Cas nuclease linked to an engineered phloem mobility element is provided to the plant, wherein the guide RNA linked to an engineered phloem mobility element and the RNA encoding the Cas nuclease linked to an engineered phloem mobility element are transported to a meristem cell of the plant, wherein the transport to the meristem cell is increased. In some embodiments, the RNA encoding the Cas nuclease is translated in the meristem cell of the plant. In some embodiments, the Cas nuclease and the guide RNA edit the genomic target in the meristem of the plant.

[0235] Another aspect of the present disclosure provides a method of providing RNA cargo comprising genome editing reagents linked to an engineered phloem mobility element to a82MF-365821254Attorney Docket No.: 16536-20022.40plant, and thereby producing a seed comprising an edited genomic target, the method comprising: delivering a guide RNA (gRNA) directed to the genomic target in a meristem cell of a parent plant by Agrobacterium-mediated delivery; and delivering a Cas nuclease to the parent plant, wherein the gRNA and the Cas nuclease are each linked to an engineered phloem mobility element, wherein the gRNA and the Cas nuclease are transported to a meristem cell of the plant; wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the parent plant, and wherein the meristem cell produces a germline cell that contributes to the seed, and thereby producing the seed comprising the edited genomic target. Also provided in the present disclosure is a method for producing a meristem cell having an edited genomic target, the method comprising: delivering a T-DNA vector carrying a gRNA linked to an engineered phloem mobility element to the roots of a plant, wherein the gRNA is transported to a meristem cell of the plant,, wherein the meristem cell comprises a Cas nuclease; allowing the gRNA and the Cas nuclease to modify the meristem cell; and thereby producing the meristem cell having the edited genomic target. In some embodiments, the edited genomic target is inherited by at least one progeny or seed of the plant. In some embodiments, the method further comprises allowing the meristem cell to generate a seed comprising the edited genomic target and collecting the seed. In some embodiments, the method further comprises growing the seed. In some embodiments, the method further comprises retrieving a progeny of the scion, wherein the progeny comprises the edited genomic target. Another aspect of the present disclosure is a seed produced by the methods herein, wherein the produced seed comprises the edited genomic target. Also provided is a meristem cell produced by the methods herein, wherein the meristem cell comprises the edited genomic target. In some embodiments of the present disclosure, the progeny does not inherit the guide RNA and / or the Cas enzyme.

[0236] In some embodiments of the present disclosure, a method for producing a meristem cell having a targeted genomic modification is provided. The method comprises delivering an Agrobacterium viral vector carrying a guide RNA linked to an engineered phloem mobility element to the root of a plant, wherein the guide RNA linked to the engineered phloem mobility element is transported to a meristem cell of the plant, wherein the meristem cell expresses a Cas enzyme; allowing the gRNA and the Cas enzyme to modify the meristem cell; and thereby producing the meristem cell having the targeted genomic modification. Also provided is a meristem cell having the modification produced by the methods herein. In some embodiments, a method for producing seed comprising a targeted genomic modification is provided. The method comprises delivering an Agrobacterium vector carrying a gRNA linked to an engineered phloem mobility element to the root of a plant, wherein the gRNA linked to the 83MF-365821254Attorney Docket No.: 16536-20022.40engineered phloem mobility element is transported in the plant to a meristem cell, wherein the meristem cell expresses a Cas nuclease; wherein the gRNA and the genomic modification enzyme modifies the meristem cell; wherein the meristem cell produces a germline that forms seed; and thereby producing seed having the targeted genomic modification. Also provided is a seed comprising an inherited modification in a gene of interest in a plant, the seed produced by the methods herein. In some embodiments, the seed does not comprise the guide RNA and / or the Cas enzyme.

[0237] The present disclosure may be used for genomic editing of any plant species, including, but not limited to, monocots and dicots (i.e., monocotyledons and dicotyledons, respectively). Examples of plant species of interest include, but are not limited to, com (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), camelina (Camelina sativa), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), quinoa (Chenopodium quinoa), chicory (Cichorium intybus), lettuce (Lactuca sativa), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Primus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oil palm (Elaeis guineensis), poplar (Populus spp.), eucalyptus (Eucalyptus spp.), oats (Avena sativa), barley (Hordeum vulgare), sesame (Sesamum spp.), flax (Linum usitatissimum), cannabis (Cannabis spp.), a vegetable crop, a forage crop, an industrial crop, a woody crop, a biomass crop, an ornamental, and a conifer.

[0238] In some embodiments, the graft is a heterograft. In other embodiments, the graft is a homograft. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and / or rootstock is a dicot. In some embodiments, the scion and / or rootstock is a monocot. In some embodiments, the scion is soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana, or wheat.84MF-365821254Attorney Docket No.: 16536-20022.40

[0239] In some embodiments, the meristem is edited. In some embodiments, the genome of a meristem of a plant scion grafted onto a rootstock is edited. In some embodiments, the meristem cell is edited. In some embodiments, the genome of a meristem cell of a plant scion grafted onto a rootstock is edited.V. DeliveryA. Vectors

[0240] Vectors are used to deliver nucleic acids to plant cells. In some embodiments, the vector is capable of autonomous replication within the host cell. In other embodiments, the vector is integrated into the genome of the host cell and replicated with the host genome. In some embodiments, termed “expression vectors”, the genes of the vector are expressed or are capable of being expressed under certain conditions. In some embodiments, the vector contains one or more regulatory elements linked to a gene. In some embodiments, the vector contains a promoter. In some embodiments, the promoter is a constitutive promoter, a conditional promoter, an inducible promoter, or a temporally or spatially specific promoter (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter). In some embodiments, a vector is introduced to a host cell to produce RNA transcripts, proteins, or peptides within the host cell, as encoded by the contained nucleic acid.

[0241] In some embodiments of the method, the nucleic acid described herein can contained within any suitable plant transformation plasmid or vector. In some embodiments, the plant transformation plasmid or vector further comprises a selectable or screenable marker, such as but not limited to a fluorescent protein or an herbicide -resistance protein. In some embodiments, the recombinant plant virus provided herein further comprises an expression cassette comprising an endogenous visible marker gene or a reporter gene, optionally wherein the reporter gene encodes a fluorescent reporter.

[0242] In some embodiments, a T-DNA vector is used to deliver at least one nucleic acid to plant cells. In some embodiments, a T-DNA binary vector is used. In some embodiments, a T-DNA superbinary vector system is used. In other embodiments, a T-DNA ternary vector system is used. In some embodiments, the T-DNA system further comprises an additional virulence gene cluster. In some embodiments, the T-DNA system further comprises an accessory plasmid or virulence helper plasmid. In some embodiments, the T-DNA vector is an Agrobacterium vector. In some embodiments, the T-DNA vector comprises a hairy root system.85MF-365821254Attorney Docket No.: 16536-20022.40

[0243] In some embodiments, the T-DNA vector is an Agrobacterium rhizogenes vector. Agrobacterium rhizogenes, also known as Rhizobium rhizogenes, is a gram-negative soil bacteria that is capable of infecting the roots of a variety of plant species. Transformation of cells of the plant root with the Ri (root inducing) plasmid of the bacteria results in random integration of the genes from the Ri plasmid into the plant cell genome. This leads to expression of the genes from the Ri plasmid in the cells of the root, resulting in the host plant producing branching root overgrowth at the site of infection in what is known as “hairy root syndrome”. Replacement of the genes of the Ri plasmid with the desired transformation product, while maintaining the virulence genes, results in the ability to produce transgenic roots that are express the genes of the desired transformation product.

[0244] In some embodiments, the nucleic acid encoding the RNA cargo is a T-DNA vector. In some embodiments, more than one RNA cargo is provided to the plant. In some embodiments, the more than one RNA cargo is provided in the same vector. In some embodiments, the more than one RNA cargo is provided in different vectors. In some embodiments, the RNA cargo comprises a reporter. In some embodiments, the RNA cargo comprises a Cas nuclease and / or a guide RNA (gRNA) for the Cas nuclease.

[0245] In some embodiments, the nucleic acid encoding the Cas nuclease and the nucleic acid encoding the guide RNA are provided in the same vector. In some embodiments, the nucleic acid encoding the Cas nuclease and the nucleic acid encoding the guide RNA are provided in different vectors. In some embodiments, the vector is a T-DNA vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral vector or a T-DNA vector.

[0246] In some embodiments of the present disclosure, provided is a method of editing a genomic target in a meristem cell a plant, the method comprising delivering a guide RNA (gRNA) directed to the genomic target to the root of the plant by a vector; and delivering a Cas nuclease to the plant, wherein the Cas nuclease and / or the guide RNA are linked to an engineered phloem mobility element and are transported within the plant to a meristem cell of the plant, wherein the Cas nuclease and the guide RNA edit the genomic target in the meristem cell of the plant, thereby editing the genomic target in the meristem cell. In some embodiments of the method, the gRNA and / or the Cas nuclease linked to an engineered phloem mobility element is contained within a T-DNA vector. In some embodiments, the gRNA and / or the Cas nuclease linked to an engineered phloem mobility element is contained within an Agrobacterium rhizogenes vector.86MF-365821254Attorney Docket No.: 16536-20022.40

[0247] In some embodiments, a first RNA cargo linked to an engineered phloem mobility element is delivered through an Agrobacterium rhizogenes T-DNA vector, and a second RNA cargo linked to an engineered phloem mobility element is delivered via at least one viral vector, including comoviruses. Suitable comovirus vectors include, for example, a bean pod mottle virus (BPMV) vector and the like. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a positive strand RNA virus. In embodiments of the method, the second component of the engineered system or a component thereof is delivered via at least one viral vector selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, gemini viruses, begomoviruses, tobamoviruses, potexviruses, potyviruses, tobraviruses, tombusviruses, bromoviruses, carmoviruses, alfamoviruses, cucumoviruses, comoviruses, and hordeviruses. See, e.g., Peyret and Lomonossoff Plant Biotechnol. J. 2015, 13:1121. Suitable tobamovirus vectors include, for example, a tomato mosaic virus (ToMV) vector, a tobacco mosaic virus (TMV) vector, a tobacco mild green mosaic virus (TMGMV) vector, a pepper mild mottle virus (PMMoV) vector, a paprika mild mottle virus (PaMMV) vector, a cucumber green mottle mosaic virus (CGMMV) vector, a kyuri green mottle mosaic virus (KGMMV) vector, a hibiscus latent fort pierce virus (HLFPV) vector, an odontoglossum ringspot virus (ORSV) vector, a rehmannia mosaic virus (ReMV) vector, a Sammon's opuntia virus (SOV) vector, a wasabi mottle virus (WMoV) vector, a youcai mosaic virus (YoMV) vector, a sunn-hemp mosaic virus (SHMV) vector, and the like. Suitable Potexvirus vectors include, for example, a potato virus X (PVX) vector, a potato aucuba mosaic virus (PAMV) vector, an Alstroemeria virus X (AlsVX) vector, a cactus virus X (CVX) vector, a Cymbidium mosaic virus (CymMV) vector, a hosta virus X (HVX) vector, a lily virus X (LVX) vector, a Narcissus mosaic virus (NMV) vector, a Nerine virus X (NVX) vector, a Plantago asiatica mosaic virus (PIAMV) vector, a strawberry mild yellow edge virus (SMYEV) vector, a tulip virus X (TVX) vector, a white clover mosaic virus (WC1MV) vector, a bamboo mosaic virus (BaMV) vector, a foxtail mosaic virus (FoMV) vector, and the like. Suitable Potyvirus vectors include, for example, a wheat streak mosaic virus (WSMV), a potato virus Y (PVY) vector, a bean common mosaic virus (BCMV) vector, a clover yellow vein virus (C1YVV) vector, an East Asian Passiflora virus (EAPV) vector, a Freesia mosaic virus (FreMV) vector, a Japanese yam mosaic virus (JYMV) vector, a lettuce mosaic virus (LMV) vector, a Maize dwarf mosaic virus (MDMV) vector, an onion yellow dwarf virus (OYDV) vector, a papaya ringspot virus (PRSV) vector, a pepper mottle virus (PepMoV) vector, a Perilla mottle virus (PerMoV) vector, a plum pox virus (PPV) vector, a potato virus A (PVA) vector, a sorghum mosaic virus (SrMV) vector, a soybean mosaic virus (SMV) vector, a 87MF-365821254Attorney Docket No.: 16536-20022.40sugarcane mosaic virus (SCMV) vector, a tulip mosaic virus (TulMV) vector, a turnip mosaic virus (TuMV) vector, a watermelon mosaic virus (WMV) vector, a zucchini yellow mosaic virus (ZYMV) vector, a tobacco etch virus (TEV) vector, and the like. Suitable Tobravirus vectors include, for example, a tobacco rattle virus (TRV) vector and the like. Suitable Tombusvirus vectors include, for example, a tomato bushy stunt virus (TBSV) vector, an eggplant mottled crinkle virus (EMCV) vector, a grapevine Algerian latent virus (GALV) vector, and the like. Suitable Cucumovirus vectors include, for example, a cucumber mosaic virus (CMV) vector, a peanut stunt virus (PSV) vector, a tomato aspermy virus (TAV) vector, and the like. Suitable Bromovirus vectors include, for example, a brome mosaic virus (BMV) vector, a cowpea chlorotic mottle virus (CCMV) vector, and the like. Suitable Carmovirus vectors include, for example, a carnation mottle virus (CarMV) vector, a melon necrotic spot virus (MNSV) vector, a pea stem necrotic virus (PSNV) vector, a turnip crinkle virus (TCV) vector, and the like. Suitable Alfamovirus vectors include, for example, an alfalfa mosaic virus (AMV) vector, and the like. Suitable Comovirus vectors include, for example, a bean pod mottle virus (BPMV) vector, a cowpea mosaic virus (CPMV) vector, and the like. Suitable Hordevirus vectors include, for example, a barley stripe mosaic virus (BSMV) vector, and the like. Suitable Begomovirus vectors include, for example, a cabbage leaf curl virus (CabLCV) vector, a soybean mild mottle virus (SbMMV) vector, and the like. Suitable Geminivirus vectors include, for example, a bean yellow dwarf virus (BeYDV) vector, a beet curly top virus (BCTV) vector, a tobacco yellow dwarf virus (TYDV) vector, and the like. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a positive strand RNA virus. In some embodiments, the first RNA cargo comprises a guide RNA and the second RNA cargo comprises a Cas nuclease. In some embodiments, the Cas nuclease is delivered by virus-mediated delivery. In some embodiments, the viral vector comprises the Cas nuclease. In some embodiments, the viral vector comprising the gRNA further comprises the Cas nuclease. In some embodiments, the recombinant plant virus used in the virus-mediated delivery is a negative strand RNA virus. In some embodiments, the recombinant plant virus used in the virus-mediated delivery has a segmented genome. In some embodiments, the recombinant plant virus used in the virus-mediated delivery further comprises an expression cassette comprising a reporter gene. In some embodiments, the reporter gene encodes a fluorescent reporter. In some embodiments, the recombinant plant virus is capable of cell-to-cell movement. In some embodiments, RNA encoding the gRNA and / or the Cas nuclease is delivered to the meristem cell of the plant by transport from another plant tissue. In some embodiments, the Cas nuclease is delivered to the meristem cell of the plant in a second viral 88MF-365821254Attorney Docket No.: 16536-20022.40vector comprising the Cas nuclease. In embodiments of the method, the engineered system or a component thereof is delivered via at least one bacterial vector capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. In some embodiments, a viral vector may be delivered to a plant by transformation with Agrobacterium.i. Hairy Root Systems

[0248] In some embodiments, the T-DNA vector is an Agrobacterium rhizogenes vector. Agrobacterium rhizogenes, also known as Rhizobium rhizogenes, is a gram-negative soil bacteria that is capable of infecting the roots of a variety of plant species. Transformation of cells of the plant root with the Ri (root inducing) plasmid of the bacteria results in random integration of the genes from the Ri plasmid into the plant cell genome. This leads to expression of the genes from the Ri plasmid in the cells of the root, resulting in the host plant producing branching root overgrowth at the site of infection in what is known as “hairy root syndrome”. Replacement of the genes of the Ri plasmid with the desired transformation product, while maintaining the virulence genes, results in the ability to produce transgenic roots that are express the genes of the desired transformation product.

[0249] In some embodiments, an RNA cargo linked to an engineered phloem mobility element described herein is provided to a plant via a hairy root system. Hairy root systems occur when a naturally occurring soil bacterium Agrobacterium rhizogenes, which contains root-inducing plasmids (Ri plasmids), infects plant roots and causes them to grow abnormally and produce a food source for the bacterium opines. The roots are easy to culture in artificial media, as hormones are not needed in contrast to adventitious roots, and they are neoplastic with indefinite growth. Roots produced via A. rhizogenes infection show a high growth rate, and genetic and biochemical stability. Ri plasmids can be engineered to contain T-DNA which is used for genetic transformation of the plant cells.B. Delivery of RNA Cargo to a Plant

[0250] In some aspects, RNA cargos are RNA molecules that are linked to engineered phloem mobility elements, provided to a plant, and transported to the meristem of a plant. In some embodiments, the RNA cargo comprises mRNA. In some embodiments, the RNA cargo comprises siRNA. In some embodiments, the RNA cargo comprises gRNA. In some embodiments, the RNA cargo comprises IncRNA. In some aspects, an RNA cargo linked to an 89MF-365821254Attorney Docket No.: 16536-20022.40engineered phloem mobility element as described herein may be any RNA suitable for use in a plant.

[0251] In some embodiments, an RNA cargo may comprise a reporter, for example, a fluorophore. In some embodiments, the RNA cargo comprises an herbicide resistance cassette. In some embodiments, the RNA cargo comprises a glyphosate resistance cassette.

[0252] RNA cargos linked to engineered phloem mobility elements that are aspects of the invention can be delivered to a plant cell using various techniques and agents. In some embodiments, the plant cell is a cell of a rootstock. In some embodiments, the plant cell is a cell of a leaf. In some embodiments, the plant cell is a cell of a grafted scion. In some embodiments, the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, the plant cell is a cell of a plant cutting. In some embodiments, the plant cell is a cell of a plant cell culture. In some embodiments, the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). In embodiments, one or more treatments is employed to deliver RNA cargo (i.e., RNA cargo linked to an engineered phloem mobility element described herein) into a plant cell or plant protoplast, e.g., through barriers such as a cell wall or a plasma membrane or nuclear envelope or other lipid bilayer. In an embodiment, RNA cargo is delivered directly, for example by direct contact of the polynucleotide composition with a plant cell or plant protoplast. An RNA cargo-containing composition in the form of a lysate, a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant cell or plant protoplast (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid RNA cargo-containing composition, whereby the RNA cargo is delivered to the plant cell or plant protoplast. For example, a plant cell or plant protoplast is abrased with powder and rubbed with the composition containing RNA cargo. In embodiments, the RNA cargo-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In embodiments, the RNA cargo-containing composition is introduced into a plant cell or plant protoplast e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive 90MF-365821254Attorney Docket No.: 16536-20022.40pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in U.S. Published Patent Application 2014 / 0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the RNA cargo-containing composition to a plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In embodiments, the RNA cargo-containing composition is provided to a plant cell or plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the gRNA; see, e.g., Broothaerts et al. Nature 2005, 433: 629-633. Bacteria may be transformed by any method known in the art, including but not limited to electroporation. Any of these techniques or a combination thereof are alternatively employed on the plant part or tissue or intact plant (or seed) from which a plant cell or plant protoplast is optionally subsequently obtained or isolated; in embodiments, the RNA cargo-containing composition is delivered in a separate step after the plant cell or plant protoplast has been obtained or isolated.

[0253] In some embodiments, a treatment employed in delivery of an RNA cargo (i.e., an RNA cargo linked to an engineered phloem mobility element described herein) to a plant cell or plant protoplast is carried out under a specific thermal regime, which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal growth of the plant cell or plant protoplast occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the plant cell or plant protoplast occurs), or treating at a combination of different temperatures. In embodiments, a specific thermal regime is carried out on a plant cell or plant protoplast, or on a plant or plant part from which a plant cell or plant protoplast is subsequently obtained or isolated, in one or more steps separate from the RNA cargo delivery. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a rootstock. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a grafted scion. In some embodiments, a specific thermal regime is carried out on a 91MF-365821254Attorney Docket No.: 16536-20022.40plant cell, wherein the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cutting. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cell culture. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).

[0254] In some embodiments, a treatment employed in delivery of an RNA cargo to a plant, plant cell, or plant protoplast is carried out under a specific thermal regime, which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal growth of the plant cell or plant protoplast occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the plant cell or plant protoplast occurs), or treating at a combination of different temperatures. In embodiments, a specific thermal regime is carried out on a plant cell or plant protoplast, or on a plant or plant part from which a plant cell or plant protoplast is subsequently obtained or isolated, in one or more steps separate from the RNA cargo delivery. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a rootstock. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a grafted scion. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cutting. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cell culture. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).

[0255] In some embodiments, a treatment employed in delivery of an RNA cargo (i.e., an RNA cargo linked to an engineered phloem mobility element described herein) to a plant, plant cell, or plant protoplast is carried out under a specific light regime, which can involve various photoperiods of light and / or dark stress on a plant. In some embodiments, to kickstart 92MF-365821254Attorney Docket No.: 16536-20022.40Agrobacterium-mediated transformation upon T-DNA vector delivery, plants are additionally stressed by placing them in the dark at 20 °C for 48h (24h prior and 24h after inoculation). In some embodiments, plants are grown in a 16h day (22°C) 8h night (20°C) light regime and watered regularly beyond this stress period.

[0256] The polynucleotides, ribonucleoproteins, DNA expression systems, engineered systems, vectors, mRNAs, guide RNAs, and Cas nucleases (collectively referred to here as “RNA cargo”) that are aspects of the invention are linked to an engineered phloem mobility element described herein and can be delivered to a plant cell using various techniques and agents. In some embodiments, the plant cell is a cell of a rootstock. In some embodiments, the plant cell is a cell of a leaf. In some embodiments, the plant cell is a cell of a grafted scion. In some embodiments, the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, the plant cell is a cell of a plant cutting. In some embodiments, the plant cell is a cell of a plant cell culture. In some embodiments, the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). In some embodiments, one or more treatments is employed to deliver genome editing reagents into a plant cell or plant protoplast, e.g., through barriers such as a cell wall or a plasma membrane or nuclear envelope or other lipid bilayer. In an embodiment, RNA cargos are delivered directly, for example by direct contact of the polynucleotide composition with a plant cell or plant protoplast. A RNA cargo-containing composition in the form of a sap, a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a soybean plant cell or soybean plant protoplast (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). A RNA cargo-containing composition in the form of a lysate, a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant cell or plant protoplast (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid RNA cargo-containing composition, whereby the RNA cargo is delivered to the plant cell or plant protoplast. In 93MF-365821254Attorney Docket No.: 16536-20022.40embodiments, the RNA cargo-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In embodiments, the RNA cargo-containing composition is introduced into a plant cell or plant protoplast e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in U.S. Published Patent Application 2014 / 0287509, incorporated by reference in its entirety herein. In another example of delivery to the plant cell or plant protoplast, a plant cell or plant protoplast is abrased with powder and rubbed with the composition containing RNA cargos. Other techniques useful for delivering the RNA cargo-containing composition to a plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In embodiments, the RNA cargo-containing composition is provided to a plant cell or plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide comprising or encoding the RNA cargo linked to the engineered phloem mobility element; see, e.g., Broothaerts et al. Nature 2005, 433: 629-633. Bacteria may be transformed by any method known in the art, including but not limited to electroporation. Any of these techniques or a combination thereof are alternatively employed on the plant part or tissue or intact plant (or seed) from which a plant cell or plant protoplast is optionally subsequently obtained or isolated; in embodiments, the RNA cargo-containing composition is delivered in a separate step after the plant cell or plant protoplast has been obtained or isolated.

[0257] In some embodiments, a whole plant or plant part or seed, or an isolated plant cell or plant protoplast, or the plant or plant part from which a plant cell or plant protoplast is obtained or isolated, is treated with one or more delivery agents comprising an engineered phloem mobility element described herein which can include at least one chemical, enzymatic, or physical agent, or a combination thereof. In embodiments, an RNA cargo-containing composition further includes one or more one chemical, enzymatic, or physical agent for 94MF-365821254Attorney Docket No.: 16536-20022.40delivery, wherein the RNA cargo is linked to an engineered phloem mobility element in the RNA cargo-containing composition. In some embodiments, the treated plant cell is a cell of a rootstock. In some embodiments, the treated plant cell is a cell of a grafted scion. In some embodiments, the RNA cargo linked to the engineered phloem mobility element is delivered to the scion by transport from a grafted rootstock. In some embodiments, the methods provided herein further comprises transforming the rootstock with a nucleic acid encoding the RNA cargo prior to grafting, wherein the nucleic acid encoding the RNA cargo is linked to an engineered phloem mobility element. In some embodiments, the scion and the rootstock are the same plant species. In some embodiments, the scion and the rootstock are different plant species. In some embodiments, the rootstock is canola, alfalfa, com, oat, sorghum, sugarcane banana, or wheat. In some embodiments, the treated plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, the treated plant cell is a cell of a plant cutting. In some embodiments, the treated plant cell is a cell of a plant cell culture. In some embodiments, the treated plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). Treatment with the chemical, enzymatic or physical agent can be carried out simultaneously with the RNA cargo delivery, or in one or more separate steps that precede or follow the RNA cargo delivery. In embodiments, a chemical, enzymatic, or physical agent, or a combination of these, is associated or complexed with an RNA cargo composition; examples of such associations or complexes include those involving non-covalent interactions (e.g., ionic or electrostatic interactions, hydrophobic or hydrophilic interactions, formation of liposomes, micelles, or other heterogeneous composition) and covalent interactions (e.g., peptide bonds, bonds formed using cross-linking agents). In non-limiting examples, a genome editing reagent is provided as a liposomal complex with a cationic lipid, or as a complex with a carbon nanotube, or as a fusion protein between the nuclease and a cell-penetrating peptide. Examples of agents useful for delivering a genome editing reagent include the various cationic liposomes and polymer nanoparticles reviewed by Zhang et al. (2007) J Controlled Release, 123:1-10, and the crosslinked multilamellar liposomes described in U.S. Patent Application Publication 2014 / 0356414 Al, incorporated by reference in its entirety herein.95MF-365821254Attorney Docket No.: 16536-20022.40i. Delivery of Genome Editing Reagents as RNA Cargo

[0258] In some embodiments, the RNA cargo linked to the engineered phloem mobility element comprises one or more genome editing reagents, such as a Cas nuclease and / or a guide RNA for a Cas nuclease. Guide RNAs and Cas enzymes (collectively referred to here as “genome editing reagents”) can be linked to engineered phloem mobility elements described herein and delivered to a plant cell using various techniques and agents. The polynucleotides, ribonucleoproteins, DNA expression systems, engineered systems, and vectors (collectively referred to here as “genome editing reagents”) described herein can be linked to an engineered phloem mobility element and delivered to a plant cell using various techniques and agents. In some embodiments, the genome editing reagents linked to the engineered phloem mobility element are transported within the vascular system of the plant to the meristem of the plant. In some embodiments, the plant cell is a cell of a rootstock. In some embodiments, the plant cell is a cell of a leaf. In some embodiments, the plant cell is a cell of a grafted scion. In some embodiments, the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, the plant cell is a cell of a plant cutting. In some embodiments, the plant cell is a cell of a plant cell culture. In some embodiments, the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). In embodiments, one or more treatments is employed to deliver genome editing reagents into a plant cell or plant protoplast, e.g., through barriers such as a cell wall or a plasma membrane or nuclear envelope or other lipid bilayer. In an embodiment, genome editing reagents are delivered directly, for example by direct contact of the polynucleotide composition with a plant cell or plant protoplast. A genome editing reagent-containing composition in the form of a lysate, a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant cell or plant protoplast (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid genome editing reagent-containing composition, whereby the genome editing reagent is delivered to the plant cell or plant protoplast. For example, a plant cell or plant protoplast is abrased with powder and rubbed with the composition containing genome editing reagents. In embodiments,96MF-365821254Attorney Docket No.: 16536-20022.40the genome editing reagent-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In embodiments, the genome editing reagent-containing composition is introduced into a plant cell or plant protoplast e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in U.S. Published Patent Application 2014 / 0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the genome editing reagent-containing composition to a plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In embodiments, the genome editing reagent-containing composition is provided to a plant cell or plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the gRNA; see, e.g., Broothaerts et al. Nature 2005, 433: 629-633. Bacteria may be transformed by any method known in the art, including but not limited to electroporation. Any of these techniques or a combination thereof are alternatively employed on the plant part or tissue or intact plant (or seed) from which a plant cell or plant protoplast is optionally subsequently obtained or isolated; in embodiments, the genome editing reagent-containing composition is delivered in a separate step after the plant cell or plant protoplast has been obtained or isolated.In some embodiments, the RNA cargo comprises a genome editing reagent, and a treatment employed in delivery of a genome editing reagent linked to an engineered phloem element described herein to a plant cell or plant protoplast is carried out under a specific thermal regime, which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal growth of the plant cell or plant protoplast occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the plant cell or plant protoplast occurs), or treating at a combination of different temperatures. In embodiments, a specific thermal regime is carried out on a plant cell or plant protoplast, or on 97MF-365821254Attorney Docket No.: 16536-20022.40a plant or plant part from which a plant cell or plant protoplast is subsequently obtained or isolated, in one or more steps separate from the genome editing reagent delivery. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a rootstock. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a grafted scion. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a seed (including mature seed and immature seed). In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cutting. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant cell culture. In some embodiments, a specific thermal regime is carried out on a plant cell, wherein the plant cell is a cell of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants).

[0259] Compositions comprising: (i) RNA molecules comprising an engineered phloem mobility element linked to an RNA cargo comprising nucleic acid encoding Cas nuclease and / or guide RNA(s); (ii) an Agrobacterium rhizogenes vector comprising or encoding an engineered phloem mobility element linked to an RNA cargo comprising nucleic acid encoding Cas nuclease and / or guide RNA(s); and / or (iii) donor DNA templates can further comprise components that include:(a) solvents (e.g., water, dimethylsulfoxide, dimethylformamide, acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide, alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and other solvents miscible or emulsifiable with water or that will dissolve phosphonucleotides in non-aqueous systems);(b) fluorocarbons (e.g., perfluorodecalin, perfluoromethyldecalin);(c) glycols or polyols (e.g., propylene glycol, polyethylene glycol);(d) surfactants, including cationic surfactants, anionic surfactants, non-ionic surfactants, and amphiphilic surfactants, e.g., alkyl or aryl sulfates, phosphates, sulfonates, or carboxylates; primary, secondary, or tertiary amines; quaternary ammonium salts; sultaines, betaines; cationic lipids; phospholipids; tallow amine; bile acids such as cholic acid; saponins or glycosylated triterpenoids or glycosylated sterols (e.g., saponin commercially available as catalogue number 47036-50g-F, Sigma-Aldrich, St. Louis, MO); long chain alcohols; organosilicone surfactants including nonionic organosilicone surfactants such as trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene 98MF-365821254Attorney Docket No.: 16536-20022.40oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as SIL WET L-77TM brand surfactant having CAS Number 27306-78-1 and EPA Number CAL. REG. NO. 5905-50073-AA, Momentive Performance Materials, Inc., Albany, N.Y.); specific examples of useful surfactants include sodium lauryl sulfate, the Tween series of surfactants, Triton-XlOO, Triton-X114, CHAPS and CHAPSO, Tergitol-type NP-40, and Nonidet P-40;(e) lipids, lipoproteins, lipopolysaccharides;(f) acids, bases, caustic agents; buffers;(g) peptides, proteins, or enzymes (e.g., cellulase, pectolyase, maceroenzyme, pectinase), including cell-penetrating or pore-forming peptides (e. g., (B0100)2K8, Genscript; polylysine, poly-arginine, or poly-homoarginine peptides; gamma zein, see US Patent Application publication 2011 / 0247100, incorporated herein by reference in its entirety; transcription activator of human immunodeficiency virus type 1 (“HIV-1 Tat”) and other Tat proteins, see, e. g., www[dot]lifetein[dot]com / Cell_Penetrating_Peptides[dot]html and Jarver Mol. Therapy-Nucleic Acids 2012, 1: e27,l - 17); octa-arginine or nona-arginine; poly-homoarginine (see Unnamalai et al. FEBS Letters 2004, 566: 307 - 310); see also the database of cell-penetrating peptides CPPsite 2.0 publicly available at webs[dot]iiitd[dot]edu[dot]in / Raghava / cppsite (Kardani and Bolhassani J Mol Biol 2021, 433(11): 166703)(h) RNase inhibitors;(i) cationic branched or linear polymers such as chitosan, poly-lysine, DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”, e. g., PEI, branched, MW 25,000, CAS# 9002-98-6; PEI, linear, MW 5000, CAS# 9002-98-6; PEI linear, MW 2500, CAS# 9002-98-6);j) dendrimers (see, e. g., US Patent Application Publication 2011 / 0093982, incorporated herein by reference in its entirety);(k) counter-ions, amines or polyamines (e. g., spermine, spermidine, putrescine), osmolytes, buffers, and salts (e. g., calcium phosphate, ammonium phosphate);(l) polynucleotides (e. g., non-specific double- stranded DNA, salmon sperm DNA);(m) transfection agents (e. g., Lipofectin®, Lipofectamine®, and Oligofectamine®, and Invivofectamine® (all from Thermo Fisher Scientific, Waltham, MA), PepFect (see Ezzat et al. Nucleic Acids Res. 2011, 39: 5284 - 5298), Transit® transfection reagents (Mirus Bio, LLC, Madison, WI), and poly-lysine, poly-homoarginine, and poly-arginine molecules99MF-365821254Attorney Docket No.: 16536-20022.40including octo-arginine and nono-arginine as described in Lu et al. J. Agric. Food Chem. 2010, 58: 2288 - 2294);(n) antibiotics, including non-specific DNA double- strand-break-inducing agents (e. g., phleomycin, bleomycin, talisomycin);(o) antioxidants (e. g., glutathione, dithiothreitol, ascorbate); and / or(p) chelating agents (e. g., EDTA, EGTA).

[0260] In embodiments, the chemical agent is provided simultaneously with the RNA cargo comprising the genome editing reagent. In embodiments, the genome editing reagent is covalently or non-covalently linked or complexed with one or more chemical agent; for example, a polynucleotide genome editing reagent can be covalently linked to a peptide or protein (e.g., a cell-penetrating peptide or a pore-forming peptide) or non-covalently complexed with cationic lipids, polycations (e.g., polyamines), or cationic polymers (e.g., PEI). In embodiments, the genome editing reagent is complexed with one or more chemical agents to form, e.g., a solution, liposome, micelle, emulsion, reverse emulsion, suspension, colloid, or gel.

[0261] In embodiments, the physical agent is at least one selected from the group consisting of particles or nanoparticles (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics) in various size ranges and shapes, magnetic particles or nanoparticles (e.g., silenceMag Magnetotransfection™ agent, OZ Biosciences, San Diego, Calif.), abrasive or scarifying agents, needles or microneedles, matrices, and grids. In embodiments, particulates and nanoparticulates are useful in delivery of the polynucleotide composition or the nuclease or both. Useful particulates and nanoparticles include those made of metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., aluminum oxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polydiacetylene, and poly(3,4-ethylenedioxythiophene) hydrate), semiconductors (e.g., quantum dots), silicon (e.g., silicon carbide), carbon (e.g., graphite, graphene, graphene oxide, or carbon nanosheets, nanocomplexes, or nanotubes), and composites (e.g., polyvinylcarbazole / graphene, polystyrene / graphene, platinum / graphene, palladium / graphene nanocomposites). In embodiments, such particulates and nanoparticulates are further covalently or non-covalently functionalized, or further include modifiers or cross-linked materials such as polymers (e.g., linear or branched polyethylenimine, poly-lysine), polynucleotides (e.g., DNA or RNA), polysaccharides, lipids, polyglycols (e.g., polyethylene glycol, thiolated polyethylene glycol), polypeptides or proteins, and detectable labels (e.g., a fluorophore, an antigen, an antibody, or a quantum dot). In various embodiments, such 100MF-365821254Attorney Docket No.: 16536-20022.40particulates and nanoparticles are neutral, or carry a positive charge, or carry a negative charge. Embodiments of compositions including particulates include those formulated, e.g., as liquids, colloids, dispersions, suspensions, aerosols, gels, and solids. Embodiments include nanoparticles affixed to a surface or support, e.g., an array of carbon nanotubes vertically aligned on a silicon or copper wafer substrate. Embodiments include polynucleotide compositions including particulates (e.g., gold or tungsten or magnetic particles) delivered by a Biolistic-type technique or with magnetic force. The size of the particles used in Biolistics is generally in the “microparticle” range, for example, gold microcarriers in the 0.6, 1.0, and 1.6 micrometer size ranges (see, e.g., instruction manual for the Helios® Gene Gun System, BioRad, Hercules, Calif.; Randolph- Anderson et al. (2015) “Sub-micron gold particles are superior to larger particles for efficient Biolistic® transformation of organelles and some cell types”, Bio-Rad US / EG Bulletin 2015), but successful Biolistics delivery using larger (40 nanometer) nanoparticles has been reported in cultured animal cells; see O'Brian and Lummis (2011) BMC Biotechnol., 11:66-71. Other embodiments of useful particulates are nanoparticles, which are generally in the nanometer (nm) size range or less than 1 micrometer, e.g., with a diameter of less than about 1 nm, less than about 3 nm, less than about 5 nm, less than about 10 nm, less than about 20 nm, less than about 40 nm, less than about 60 nm, less than about 80 nm, and less than about 100 nm. Specific, non-limiting embodiments of nanoparticles commercially available (all from Sigma-Aldrich Corp., St. Louis, Mo.) include gold nanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticles with particle sizes of 10, 20, 40, 60, or 100 nm; palladium “nanopowder” of less than 25 nm particle size; single-, double-, and multiwalled carbon nanotubes, e.g., with diameters of 0.7-1.1, 1.3-2.3, 0.7-0.9, or 0.7-1.3 nm, or with nanotube bundle dimensions of 2-10 nm by 1-5 micrometers, 6-9 nm by 5 micrometers, 7-15 nm by 0.5-10 micrometers, 7-12 nm by 0.5-10 micrometers, 110-170 nm by 5-9 micrometers, 6-13 nm by 2.5-20 micrometers. Embodiments include genome editing reagentcontaining compositions including materials such as gold, silicon, cerium, or carbon, e.g., gold or gold-coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin / silica nanoparticles, nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs) such as single-, double-, or multi-walled carbon nanotubes and their chemically functionalized versions (e.g., carbon nanotubes functionalized with amide, amino, carboxylic acid, sulfonic acid, or polyethylene glycol moieties), and graphene or graphene oxide or graphene complexes; see, for example, Wong et al. (2016) Nano Lett., 16:1161-1172; Giraldo et al. (2014) Nature Materials, 13:400-409; Shen et al. (2012) Theranostics, 2:283-294; Kim et al. (2011) Bioconjugate Chem., 22:2558-2567; Wang et al. (2010) J. Am. Chem. Soc.101MF-365821254Attorney Docket No.: 16536-20022.40Comm., 132:9274-9276; Zhao et al. (2016) Nanoscale Res. Lett., 11:195-203; and Choi et al. (2016) J. Controlled Release, 235:222-235. See also, for example, the various types of particles and nanoparticles, their preparation, and methods for their use, e.g., in delivering polynucleotides and polypeptides to cells, disclosed in U.S. Patent Application Publications 2010 / 0311168, 2012 / 0023619, 2012 / 0244569, 2013 / 0145488, 2013 / 0185823, 2014 / 0096284, 2015 / 0040268, 2015 / 0047074, and 2015 / 0208663, all of which are incorporated herein by reference in their entirety.

[0262] In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more RNA molecules in one or more compositions where at least one of the RNA molecules comprises a guide RNA linked to an engineered phloem mobility element. In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more viral vectors carrying a guide RNA. In some embodiments, the guide RNA is linked to a viral vector. In some embodiments, the guide RNA is linked to a recombinant plant virus. In some embodiments, the viral vector carrying the guide RNA linked to the engineered phloem mobility element is present in a composition that contacts a plant. In some embodiments, the vector carrying the guide RNA linked to an engineered phloem mobility element is present in a composition that contacts a plant. In some embodiments, the composition contacts a seed (including mature seed and immature seed). In some embodiments, the composition contacts a plant cutting. In some embodiments, the composition contacts a plant cell culture.

[0263] In some embodiments, the methods herein further comprise infecting the plant with a plurality of viral vectors, wherein each viral vector comprises one or more gRNA and / or the Cas nuclease. In some embodiments, the plurality of viral vectors co-infect the plant simultaneously or infect the plant in more than one round of infection. In some embodiments, the composition contacts a rootstock. In some embodiments, the composition contacts a grafted scion. In some embodiments, the composition contacts a seed (including mature seed and immature seed). In some embodiments, the composition contacts a plant cutting. In some embodiments, the composition contacts a plant cell culture. In some embodiments, the composition contacts a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more RNA molecules in one or more compositions where at least one of the RNA molecules comprises an RNA encoding a 102MF-365821254Attorney Docket No.: 16536-20022.40Cas nuclease linked to an engineered phloem mobility element. In some embodiments, the composition contacts a plant cell culture. In some embodiments, the composition contacts a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, root, flowers, fruits, shoots, and explants). In certain embodiments, plants are contacted either simultaneously or sequentially with one, two, three or more viral vectors in one or more compositions where at least one of the viral vectors or viral vector systems comprises an RNA encoding a Cas nuclease, wherein the Cas is linked to an engineered phloem mobility element. In certain embodiments, one of the RNA molecules comprises a guide RNA linked to an engineered phloem mobility element and a second RNA molecule comprises RNA encoding an RNA guided Cas nuclease and is optionally linked to an engineered phloem mobility element, where the RNA guided Cas nuclease can process the RNA comprising the guide RNA to release a functional guide RNA. In certain embodiments, one of the RNA molecules comprises at least one guide RNA linked to an engineered phloem mobility element and a second RNA molecule comprises RNA encoding an RNA guided nuclease linked to an engineered phloem mobility element, where the RNA guided nuclease cannot process the RNA comprising the guide RNA to release a functional guide RNA (e.g., processing elements present in the RNA molecule comprising the gRNA linked to the engineered phloem mobility element are not recognized by the RNA-guided nuclease). In certain embodiments, guide RNAs of the first and second RNA molecule are flanked by or comprise processing elements (e.g., DRs) which are processed by different RNA-guided nuclease (e.g., a Cas 12a nuclease can process the first RNA molecule and a Casl2j nuclease can process the second RNA molecule). In certain embodiments, the guide RNA(s) of the first RNA molecule distinct from the guide RNA(s) of the second RNA molecule. Such distinct gRNAs provided by the first RNA molecule can provide for genome editing at one or more first genomic sites in a meristem cell while the distinct gRNAs provided by the second RNA molecule can provide for genome editing at one or more second genomic sites in a meristem cell. Such contacting the plant with RNA molecules in a composition can occur sequentially such that the first gRNA(s) are delivered, allowed sufficient time (e.g., about 6, 12, 18, or 20 to about 24, 30, or 36 hours) to effect desired genome edits, followed by contacting the plant with the second RNA molecules in a second composition to deliver the second gRNA(s) to effect additional desired genome edits, where such desired genome edits are effected by providing the gRNA(s) and an RNA guided nuclease in at least the meristem cell. In certain embodiments, the guide RNA(s) delivered by the first viral vector is distinct 103MF-365821254Attorney Docket No.: 16536-20022.40from the guide RNA(s) delivered by the second viral vector. Such distinct gRNAs provided by the first viral vector can provide for genome editing at one or more first genomic sites in a meristem cell while the distinct gRNAs provided by the second viral vector can provide for genome editing at one or more second genomic sites in a meristem cell. Such contacting the plant with viral vectors in a composition can occur sequentially such that the first gRNA(s) are delivered, allowed sufficient time (e.g., about 6, 12, 18, or 20 to about 24, 30, or 36 hours) to effect desired genome edi...

Claims

1. Attorney Docket No.: 16536-20022.40CLAIMSWhat is claimed is:

1. An engineered phloem mobility element comprising an RNA having the secondary structure selected from the group consisting of:>>[Structure IV]when linked to an RNA cargo, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

2. An engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising:a) a first stem of 9 nucleotides,b) a first stem loop structure comprising i) a second stem of five nucleotides comprising a bulge comprising one unpaired nucleotide and ii) a first loop comprising four nucleotides,c) a second stem loop structure comprising i) a third stem of five nucleotides and ii) a second loop comprising seven nucleotides, andd) a third stem loop structure comprising i) a third stem of four nucleotides and ii) a third loop comprising four nucleotides,further comprising:e) at least five unpaired nucleotides 5’ of the first stem,f) two unpaired nucleotides between the first stem and the first stem loop structure, g) one unpaired nucleotide between the second stem loop structure and the third stem loop structure,h) eight unpaired nucleotides between the third stem loop structure and the stem, and i) at least four unpaired nucleotides 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.165MF-365821254Attorney Docket No.: 16536-20022.

403. An engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising:a) a first stem of eight nucleotides,b) a first stem loop structure comprising i) a second stem of twelve nucleotides, comprising a first bulge comprising one unpaired nucleotide, a second bulge comprising one unpaired nucleotide, a third bulge comprising one unpaired nucleotide, and a first loop comprising six unpaired nucleotides, and ii) a second loop comprising seven nucleotides; and c) a second stem loop structure comprising i) a third stem of three nucleotides and ii) a third loop comprising seven nucleotides,further comprising:d) at least six unpaired nucleotides 5’ of the first stem,e) one unpaired nucleotide between the first stem loop structure and the second stem loop structure,f) one unpaired nucleotide between the second stem loop structure and the first stem, andg) at least two unpaired nucleotides 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

4. An engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising:a) a first stem of six nucleotides,b) a first stem loop structure comprising i) a second stem of four nucleotides, and ii) a first loop comprising nine nucleotides;c) a second stem loop structure comprising i) a third stem of five nucleotides, and ii) a second loop comprising seven nucleotides; andd) a third stem loop structure comprising i) a fourth stem of six nucleotides, and ii) a third loop comprising five nucleotides,further comprising:e) three unpaired nucleotides between the first stem and the first stem loop structure, f) one unpaired nucleotide between the first stem loop structure and the second stem loop structure,g) five unpaired nucleotides between the second stem loop structure and the third stem loop structure,166MF-365821254Attorney Docket No.: 16536-20022.40h) one unpaired nucleotides between the third stem loop structure and the first stem, andi) at least one unpaired nucleotide 3’ of the first stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

5. An engineered phloem mobility element, wherein the engineered phloem mobility element is an RNA comprising a secondary structure comprising:a) a first stem of five nucleotides, comprising a first bulge comprising one unpaired nucleotide and a second bulge comprising one unpaired nucleotide;b) a first stem loop structure comprising i) a second stem of nine nucleotides, comprising a first bulge comprising three unpaired nucleotides, a second bulge comprising two unpaired nucleotides, a third bulge comprising two unpaired nucleotides, and a fourth bulge comprising three unpaired nucleotides, and ii) a first loop comprising four nucleotides; andc) a second stem loop structure comprising i) a third stem of eight nucleotides, comprising a first bulge comprising one unpaired nucleotide and a second bulge comprising one unpaired nucleotide, and ii) a second loop comprising four nucleotides,further comprising:d) three unpaired nucleotides between the first stem and the first stem loop structure, e) four unpaired nucleotides between the first stem loop structure and the second stem loop structure,f) one unpaired nucleotide between the second stem loop structure and the stem, and g) at least one unpaired nucleotide 3’ of the stem, wherein the RNA comprises a sequence that is not a naturally occuring sequence in the plant.

6. The engineered phloem mobility element of any one of claims 1-5, wherein the engineered phloem mobility element comprises between 70-85 nucleotides, optionally wherein the engineered phloem mobility element comprises between 74-82 nucleotides.

7. The engineered phloem mobility element of any one of claims 1-6 wherein the engineered phloem mobility element folds into the secondary structure with an energy partition function167MF-365821254Attorney Docket No.: 16536-20022.40of less than -25 EpF, optionally wherein the energy partition function is less than -30 EpF, optionally wherein the energy partition function is less than -35 EpF.

8. The engineered phloem mobility element of any one of claims 1-7, wherein the engineered phloem mobility element:(a) comprises a secondary structure of> [Structure 1], and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 1-595;(b) comprises a secondary structure of> (((((((((((((•((((((•( )•)))))) )))))•((( )))•))))))))•• [Structure 2], and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 596-655,(c) comprises a secondary structure of((((((•••(((( ))))•((((( ))))) (((((( ))))))•))))))• [Structure 3], and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 656-690, or(d) comprises a secondary structure of[Structure 4], and comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to a sequence selected from the group consisting of SEQ ID NOs: 691-752.

9. The engineered phloem mobility element of any one of claims 1-8, wherein the engineered phloem mobility element comprises a sequence selected from SEQ ID NOs: 370, 593, 648, 653, 688, and 689.

10. The engineered phloem mobility element of any one of claims 1-9, wherein transport of the linked RNA cargo within the plant is increased compared to transport of the RNA cargo without the engineered phloem mobility element.168MF-365821254Attorney Docket No.: 16536-20022.4011. An RNA molecule comprising the engineered phloem mobility element of any one of claims 1-10 linked to the RNA cargo, optionally wherein the engineered phloem mobility element i) is located 3’ to the RNA cargo or ii) is located 5’ to the RNA cargo.

12. The RNA molecule of claim 11, wherein the RNA cargo:a) is an mRNA molecule,b) encodes a Cas and / or comprises a guide RNA (gRNA); and / orc) encodes a reporter, optionally a fluorescent reporter, optionally wherein the reporter is a GFP or an eGFP variant.

13. A nucleic acid encoding the RNA molecule of claim 11 or 12, optionally wherein the RNA cargo is mRNA.

14. A vector comprising the nucleic acid of claim 13.

15. A plant comprising the RNA molecule of claim 12 or the nucleic acid of claim 13.

16. The plant of claim 15, wherein the plant is selected from soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana, cotton, or wheat.

17. A method of transporting an RNA cargo to a meristem cell of a plant comprising providing to the plant the RNA cargo linked to the engineered phloem mobility element of any one of claims 1-10.

18. The method of claim 17, wherein the RNA cargo linked to the engineered phloem mobility element is delivered to the plant via a T-DNA vector, optionally wherein the T-DNA vector is delivered to a root of the plant, optionally wherein the T-DNA vector comprises a Ri plasmid from Agrobacterium rhizogenes, optionally wherein the RNA cargo linked to the phloem mobility element is delivered to the root of the plant by an Agrobacterium rhizogenes transformation, optionally wherein the Agrobacterium rhizogenes transformation produces transgenic hairy roots, and optionally wherein the meristem cell is in a shoot apical meristem or an axillary meristem.169MF-365821254Attorney Docket No.: 16536-20022.4019. The method of claim 17 or 18, wherein the RNA cargo linked to the engineered phloem mobility element is delivered to a root of the plant by one or more of:i) injecting a composition comprising the RNA cargo linked to the engineered phloem mobility element into the root;ii) incubating the root with a composition comprising the RNA cargo linked to the engineered phloem mobility element; andiii) an Agrobacterium rhizogenes transformation.

20. The method of claim 19, wherein the RNA cargo comprises nucleic acid encoding a Cas nuclease, optionally wherein a nucleic acid encoding a gRNA for the Cas nuclease is provided separately to the plant, optionally wherein the nucleic acid encoding the gRNA for the Cas nuclease is linked to a second phloem mobility element, optionally wherein:a) the second phloem mobility element is an engineered phloem mobility element of any one of claims 1-10, orb) the second phloem mobility element comprises or is derived from:i) a Flower Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); orii) an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.

21. The method of claim 19 or 20, wherein the RNA cargo comprises nucleic acid encoding a gRNA for a Cas nuclease, optionally wherein the Cas nuclease is constitutively expressed in the plant or in the roots of the plant22. The method of claim 21, wherein the plant comprises a rootstock and a scion grafted onto the rootstock, optionally wherein nucleic acid encoding the Cas nuclease is expressed in the rootstock, wherein the nucleic acid encoding the Cas nuclease is linked to a second phloem mobility element, and optionally whereina) the second phloem mobility element is an engineered phloem mobility element of any one of claims 1-10, orb) the second phloem mobility element comprises or is derived from:170MF-365821254Attorney Docket No.: 16536-20022.40i) a Flower Locus T (FT)-derived sequence, a tRNA like sequence (TLS), a meristem transport component (MTC); orii) an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides, and a variable loop.

23. The method of any one of claims 17-19, wherein the RNA cargo comprises i) nucleic acid encoding a Cas nuclease and ii) a gRNA for the Cas nuclease.

24. The method of claim 20 or 23, wherein the nucleic acid encoding the Cas nuclease is transported to the meristem, wherein the Cas nuclease is translated in the meristem.

25. The method of any one of claims 20-24, wherein a genomic target within a cell in the meristem is edited.

26. The method of any one of claims 20-25, wherein the plant comprises a rootstock and a scion grafted onto the rootstock, optionallyi) wherein the scion and the rootstock are different plant species, orii) wherein the scion and the rootstock are the same plant species, and optionally a) wherein the scion and / or rootstock is a dicot, orb) wherein the scion and / or rootstock is a monocot.

27. The method of any one of claims 17-26, wherein the plant is a dicot.

28. The method of any one of claims 17-26, wherein the plant is a monocot.

29. The method of any one of claims 26-28, wherein the rootstock and / or scion, or plant is soy, canola, alfalfa, com, oat, sorghum, sugarcane, banana, or wheat.

30. The method of any one of claims 20, 23, or 24, wherein:(i) the nucleic acid encoding the Cas nuclease is codon-optimized for expression in dicots, optionally wherein the nucleic acid encoding the Cas nuclease is codon-optimized for expression in soybean;(ii) the nucleic acid encoding the Cas nuclease is codon-optimized for expression in monocots; or171MF-365821254Attorney Docket No.: 16536-20022.40(iii) the nucleic acid encoding the Cas nuclease is codon-optimized for expression in com, soy, or wheat.

31. The method of any one of claims 20-30, further comprising retrieving a progeny of the plant, wherein the progeny has an altered genome.

32. The method of any one of claims 20-31, wherein the guide RNA further comprises:(a) one or more modified nucleotides within five nucleotides from the 5’ end of the guide RNA; or(b) one or more modified nucleotides within five nucleotides from the 3’ end of the guide RNA; or(c) both (a) and (b);wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar, optionally(i) wherein each of the one or more modified nucleotides is independently selected from the group consisting of 2’-O-methyl nucleotide, a 2’-0-methyl-3’phosphorothioate nucleotide, a 2’-0-methyl-3’phosphonoacetate nucleotide, and a 2’-O-methyl-3’-phosphonothioacetate nucleotide, or(ii) wherein the one or more modified nucleotides comprises a modified internucleotide linkage or a modified terminal phosphate group selected from the group consisting of an alkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, a boranophosphonate, a phosphorothioate, a phosphonothioacetate, and a phosphorodithioate group.

33. The method of any one of claims 20-32, wherein the method further comprises delivering a donor template DNA to the plant, optionally wherein the sequence from the donor template DNA is incorporated into the genome of the plant at the genomic target.

34. The method of any one of claims 20-33, wherein the editing of the genomic target results in the increased expression of a gene of interest in the plant, wherein the genomic target inhibits the gene of interest when expressed in a control plant, optionally wherein the genomic target is involved in viral defense, Non-Homologous End Joining (NHEJ), Mismatch Repair (MMR), or condensing chromatin.172MF-365821254Attorney Docket No.: 16536-20022.4035. The method of any one of claims 20-34, the method further comprising screening the plant for successful editing of the genomic target, said screening comprising:(a) visually assessing the plant for at least one desired phenotype; and / or(b) sequencing nucleic acid of cells produced by the meristem cell.

36. A plant, seed, or meristem cell produced by the method of claim 25, wherein the produced plant, seed, or meristem cell comprises the edited genomic target.

37. A kit comprising the viral vector system of claim 14 and an instruction manual for using the kit.

38. A method for assessing the ability of a candidate engineered phloem mobility element to promote mobility of an RNA cargo within a plant, comprising:a) delivering the candidate engineered phloem mobility element linked to the RNA cargo to the roots of the plant via an Agrobacterium rhizogenes vector,wherein the engineered phloem mobility element comprises a secondary structure when linked to the RNA cargo, andb) measuring the amount of the RNA cargo in the meristem of the plant,optionally wherein the RNA cargo comprises mRNA, and optionally wherein the RNA cargo comprises a reporter system.

39. A rootstock comprising a nucleic acid encoding a Cas nuclease, wherein the nucleic acid encoding the Cas nuclease is linked to an engineered phloem mobility element comprising a secondary structure selected from the group consisting of:>>[Structure IV],wherein the engineered phloem mobility element is not a naturally occurring RNA in the plant.173MF-365821254

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